Recombinant proteins of a pakistani strain of hepatitis E and their use in diagnostic methods and vaccines

ABSTRACT

A strain of hepatitis E virus from Pakistan (SAR-55) implicated in an epidemic of enterically transmitted non-A, non-B hepatitis, now called hepatitis E, is disclosed. The invention relates to the expression of the whole structural region of SAR-55, designated open reading frame 2 (ORF-2), in a eukaryotic expression system. The expressed protein is capable of forming HEV virus-like particles which can serve as an antigen in diagnostic immunoassays and as an immunogen or vaccine to protect against infection by hepatitis E.

The present application is a continuation-in-part of U.S. applicationSer. No. 07/947,263 filed on Sep. 18, 1992 now abandoned.

FIELD OF INVENTION

The invention is in the field of hepatitis virology. More specifically,this invention relates to recombinant proteins derived from anenterically transmitted strain of hepatitis E from Pakistan, SAR-55, andto diagnostic methods and vaccine applications which employ theseproteins.

BACKGROUND OF INVENTION

Epidemics of hepatitis E, an enterically transmitted non-A/non-Bhepatitis, have been reported in Asia, Africa and Central America(Balayan, M. S. (1987), Soviet Medical Reviews, Section E, VirologyReviews, Zhdanov, O-V. M. (ed), Chur, Switzerland: Harwood AcademicPublishers, vol. 2, 235-261; Purcell, R. G., et al. (1988) in Zuckerman,A. J. (ed), “Viral Hepatitis and Liver Disease”, New York: Alan R. Liss,131-137; Bradley, D. W. (1990), British Medical Bulletin, 46:442-461;Ticehurst, J. R. (1991) in Hollinger, F. B., Lemon, S. M., Margolis, H.S. (eds): “Viral Hepatitis and Liver Disease”, Williams and Wilkins,Baltimore, 501-513). Cases of sporadic hepatitis, presumed to behepatitis E, account for up to 90% of reported hepatitis in countrieswhere hepatitis E virus (HEV) is endemic. The need for development of aserological test for the detection of anti-HEV antibodies in the sera ofinfected individuals is widely recognized in the field, but the very lowconcentration of HEV excreted from infected humans or animals made itimpossible to use such HEV as the source of antigen for serologicaltests and although limited success was reported in propagation of HEV incell culture (Huang, R. T. et al. (1992), J. Gen. Virol., 73:1143-1148),cell culture is currently too inefficient to produce the amounts ofantigen required for serological tests.

Recently, major efforts worldwide to identify viral genomic sequencesassociated with hepatitis E have resulted in the cloning of the genomesof a limited number of strains of HEV (Tam, A. W. et al. (1991),Virology, 185:120-131; Tsarev, S. A. et al. (1992), Proc. Natl. Acad.Sci. USA, 89:559-563; Fry, K. E. et al. (1992), Virus Genes, 6:173-185).Analysis of the DNA sequences have led investigators to hypothesize thatthe HEV genome is organized into three open reading frames (ORFs) and tohypothesize that these ORFs encode intact HEV proteins.

A partial DNA sequence of the genome of an HEV strain from Burma(Myanmar) is disclosed in Reyes et al., 1990, Science, 247:1335-1339.Tam et al., 1991, and Reyes et al., PCT Patent Application WO91/15603published Oct. 17, 1991 disclose the complete nucleotide sequence and adeduced amino acid sequence of the Burma strain of HEV. These authorshypothesized that three forward open reading frames (ORFs) are containedwithin the sequence of this strain.

Ichikawa et al., 1991, Microbiol. Immunol., 35:535-543, discloses theisolation of a series of clones of 240-320 nucleotides in length uponthe screening of a λgt11 expression library with sera from HEV-infectedcynomolgus monkeys. The recombinant protein expressed by one clone wasexpressed in E. coli. This fusion protein is encoded by the 3′ region ofORF-2 of the Myanmar strain of HEV.

The expression of additional proteins encoded within the 3′ region ofORF-2 of a Mexican strain of HEV and of a Burmese strain of HEV isdescribed in Yarbough et al., 1991 J. Virology, 65:5790-5797. Thisarticle describes the isolation of two cDNA clones derived from HEV.These clones encode the proteins in the 3′ region of ORF-2. The cloneswere expressed in E. coli as fusion proteins.

Purdy et al., 1992, Archives of Virology, 123:335-349, and Favorov etal., 1992, J. of Medical Virology, 36:246-250, disclose the expressionof a larger ORF-2 protein fragment from the Burma strain in E. coli.These references, as well as those previously discussed, only disclosethe expression of a portion of the ORF-2 gene using bacterial expressionsystems. Successful expression of the full-length ORF-2 protein has notbeen disclosed until the present invention.

Comparison of the genome organization and morphological structure of HEVto that of other viruses revealed that HEV is most closely related tothe caliciviruses. Of interest, the structural proteins of calicivirusesare encoded by the 3′ portion of their genome (Neil, J. D. et al. (1991)J. Virol., 65:5440-5447; and Carter, M. J. et al. (1992), J. Arch.Virol., 122:223-235) and although there is no direct evidence that the3′ terminal part of the HEV genome also encodes the structural proteins,expression of certain small portions of the 3′ genome region inbacterial cells resulted in production of proteins reactive withanti-HEV sera in ELISA and Western blots (Yarborough, et al., (1991);Ichikawa et al. (1991); Favorov et al. (1992) and Dawson, G. J. et al.(1992) J. Virol. Meth; 38:175-186). However, the function of ORF-2protein as a structural protein was not proven until the presentinvention.

The small proteins encoded by a portion of the ORF-2 gene have been usedin immunoassay to detect antibodies to HEV in animal sera. The use ofsmall bacterially expressed proteins as antigens in serologicalimmunoassays has several potential drawbacks. First, the expression ofthese small proteins in bacterial cells often results in solubilityproblems and in non-specific cross-reactivity of patients' sera with E.coli proteins when crude E. coli lysates are used as antigens inimmunoassays (Purdy et al. (1992)). Second, the use of Western blots asa first-line serological test for anti-HEV antibodies in routineepidemiology is impractical due to time and cost constraints. An ELISAusing small-peptides derived from the 3′-terminal part of the HEV genomeresulted in the detection of only 41% positives from known HEV-infectedpatients. Third, it has been shown that for many viruses, includingPicornaviridae which is the closest family to the Caliciviridae,important antigenic and immunogenic epitopes are highly conformational(Lemon, S. M. et al. (1991), in Hollinger, F. B., Lemon, S. M.,Margolis, H. S. (eds): “Viral Hepatitis and Liver Disease”, Williams andWilkins, Baltimore, 20-24). For this reason, it is believed thatexpression in a eukaryotic system of a complete ORF encoding an intactHEV gene would result in production of a protein which could formHEV-virus-like particles. Such a complete ORF protein would have animmunological structure closer to that of native capsid protein(s) thanwould the above-noted smaller proteins which represent only portions ofthe structural proteins of HEV. Therefore, these complete ORF proteinswould likely serve as a more representative antigen and a more efficientimmunogen than the currently-used smaller proteins.

SUMMARY OF INVENTION

The present invention relates to an isolated and substantially purepreparation of a human hepatitis E viral strain SAR-55.

The invention also relates to an isolated and substantially purepreparation of the genomic RNA of the human hepatitis E viral strainSAR-55.

The invention further relates to the cDNA of the human hepatitis E viralstrain SAR-55.

It is an object of this invention to provide synthetic nucleic acidsequences capable of directing production of recombinant HEV proteins,as well as equivalent natural nucleic acid sequences. Such naturalnucleic acid sequences may be isolated from a cDNA or genomic libraryfrom which the gene capable of directing synthesis of the HEV proteinsmay be identified and isolated. For purpose of this application, nucleicacid sequence refers to RNA, DNA, cDNA or any synthetic variant thereofwhich encodes for protein.

The invention further relates to a method for detection of the hepatitisE virus in biological samples based on selective amplification ofhepatitis E gene fragments utilizing primers derived from the SAR-55cDNA.

The invention also relates to the use of single-stranded antisensepoly-or oligonucleotides derived from the SAR-55 cDNA to inhibit theexpression of hepatitis E genes.

The invention also relates to isolated and substantially purified HEVproteins and variants thereof encoded by the HEV genome of SAR-55 orencoded by synthetic nucleic acid sequences and in particular torecombinant proteins encoded by at least one complete open reading frameof HEV.

The invention also relates to the method of preparing recombinant HEVproteins derived from an HEV genomic sequence by cloning the nucleicacid and inserting the cDNA into an expression vector and expressing therecombinant protein in a host cell.

The invention also relates to the use of the resultant recombinant HEVproteins as diagnostic agents and as vaccines.

The present invention also encompasses methods of detecting antibodiesspecific for hepatitis E virus in biological samples. Such methods areuseful for diagnosis of infection and disease caused by HEV, and formonitoring the progression of such disease. Such methods are also usefulfor monitoring the efficacy of therapeutic agents during the course oftreatment of HEV infection and disease in a mammal.

This invention also relates to pharmaceutical compositions for use inprevention or treatment of Hepatitis E in a mammal.

DESCRIPTION OF FIGURES

FIG. 1 shows the recombinant vector used to express the complete ORF-2protein of the genome of HEV strain SAR-55.

FIGS. 2A and 2B are sodium dodecyl sulfate-polyacrylamide gels(SDS-PAGE) in which cell lysates of insect cells infected with wild-typebaculovirus or recombinant baculovirus (containing the gene encodingORF-2) were either stained with Coomassie blue (A) or subjected toWestern blotting with serum of an HEV-infected chimp (B). In both FIGS.2A and 2B, lane 1 contains total cell lysate of noninfected SF-9 cells;lane 2 contains lysate of cells infected with wild-type baculovirus;lane 3 contains lysate of cells infected with recombinant baculovirusand lane 4 contains molecular weight markers.

FIGS. 3A, 3A′, 3A″, 3A′″ and 3B show immunoelectron micrographs (IEM) of30 and 20 nm virus-like particles respectively, which are formed as aresult of the expression of ORF-2 protein in recombinant infected insectcells.

FIG. 4 shows the results of an ELISA using as the antigen, recombinantORF-2 which was expressed from insect cells containing the gene encodingthe complete ORF-2. Serum anti-HEV antibody levels were determined atvarious times following inoculation of cynomolgus monkeys with eitherthe Mexican (Cyno-80A82, Cyno-9A97 and Cyno 83) or Pakistani (Cyno-374)strains of HEV.

FIGS. 5A-D show the results of an ELISA using as the antigen,recombinant ORF-2 which was expressed from insect cells containing thegene encoding the complete ORF-2. Serum IgG or IgM anti-HEV levels weredetermined over time following inoculation of two chimpanzees with HEV.

FIGS. 6A-J show a comparison of ELISA data obtained using as the antigenthe recombinant complete ORF-2 protein derived from SAR-55 as theantigen vs. a recombinant partial ORF-2 protein derived from the Burmastrain of HEV (Genelabs).

FIGS. 7A-J show anti-HEV IgG ELISA and alanine aminotransferase (ALT)values for cynomolgus monkeys inoculated with ten-fold serial dilutions(indicated in parenthesis at the top of each panel) of a 10% fecalsuspension of SAR-55 HEV. Recombinant antigens used in ELISA were:glutathione-S-transferase (GST); 3-2(M), a fusion of the 3-2 epitope[Yarbough et al., (1991) J. Virol, 65:5790-5797] and GST; SG3 (B), afusion of 327 C-terminal amino acids of ORF-2 and GST [Yarbough et al.,(1993): Assay Development of diagnostic tests for Hepatitis E in“International Symposium on Viral Hepatitis and Liver Disease.Scientific Program and Abstract Volume.” Tokyo:VHFL p. 87]; and a 55 kDaORF-2 product directly expressed in insect cells.

FIGS. 8A-E show anti-HEV IgM ELISA and ALT values for positivecynomolgus monkeys inoculated with ten-fold serial dilutions (indicatedin parenthesis at the top of each panel) of the 10% fecal suspension ofSAR-55 HEV. Recombinant antigens used in ELISA were:glutathione-S-transferase (GST); 3-2(M), a fusion of the 3-2 epitope[Yarbough et al., 1991] and (GST); SG3 (B), a fusion of 327 C-terminalamino acids of ORF-2 and GST [Yarbough et al., 1993]; and the 55 kDaORF-2 product directly expressed in insect cells.

FIG. 9 shows an ethidium bromide stain of a 2% agarose gel on which PCRproducts produced from extracts of serial ten-fold dilutions (indicatedat the top of each lane of the gel) of the 10 fecal suspension of theSAR-55 HEV were separated. The predicted length of the PCR products wasabout 640 base pairs and the column marked with an (M) contains DNA sizemarkers.

FIG. 10 shows the pPIC9 vector used to express the complete ORF-2protein or lower molecular weight fragments in yeast.

DETAILED DESCRIPTION OF INVENTION

The present invention relates to an isolated and substantially purifiedstrain of hepatitis E virus (HEV) from Pakistan, SAR-55. The presentinvention also relates to the cloning of the viral genes encodingproteins of HEV and the expression of the recombinant proteins using anexpression system. More specifically, the present invention relates tothe cloning and expression of the open reading frames (ORF) of HEVderived from SAR-55.

The present invention relates to isolated HEV proteins. Preferably, theHEV proteins of the present invention are substantially homologous to,and most preferably biologically equivalent to, the native HEV proteins.By “biologically equivalent” as used throughout the specification andclaims, it is meant that the compositions are capable of formingviral-like particles and are immunogenic. The HEV proteins of thepresent invention may also stimulate the production of protectiveantibodies upon injection into a mammal that would serve to protect themammal upon challenge with a wild-type HEV. By “substantiallyhomologous” as used throughout the ensuing specification and claims, ismeant a degree of homology in the amino acid sequence to the native HEVproteins. Preferably the degree of homology is in excess of 70%,preferably in excess of 90%, with a particularly preferred group ofproteins being in excess of 99% homologous with the native HEV proteins.

Preferred HEV proteins are those proteins that are encoded by the ORFgenes. Of particular interest are proteins encoded by the ORF-2 gene ofHEV and most particularly proteins encoded by the ORF-2 gene of theSAR-55 strain of HEV. The preferred proteins of the present invention,encoded by the ORF-2 gene, form virus-like particles. The amino acidsequences of the ORF-1, ORF-2 and ORF-3 proteins are shown below as SEQID NO.: 1, SEQ ID NO.: 2, and SEQ ID NO.: 3, respectively:

                     (SEQ. ID NO.: 1) Met Glu Ala His Gln Phe Ile LysAla Pro Gly Ile Thr Thr Ala1               5                   10                   15 Ile Glu GlnAla Ala Leu Ala Ala Ala Asn Ser Ala Leu Ala Asn                20                  25                   30 Ala Val ValVal Arg Pro Phe Leu Ser His Gln Gln Ile Glu Ile                35                  40                   45 Leu Ile AsnLeu Met Gln Pro Arg Gln Leu Val Phe Arg Pro Glu                50                  55                   60 Val Phe TrpAsn His Pro Ile Gln Arg Val Ile His Asn Glu Leu                65                  70                   75 Glu Leu TyrCys Arg Ala Arg Ser Gly Arg Cys Leu Glu Ile Gly                80                  85                   90 Ala His ProArg Ser Ile Asn Asp Asn Pro Asn Val Val His Arg                95                  100                 105 Cys Phe LeuArg Pro Ala Gly Arg Asp Val Gln Arg Trp Tyr Thr                110                 115                 120 Ala Pro ThrArg Gly Pro Ala Ala Asn Cys Arg Arg Ser Ala Leu                125                 130                 135 Arg Gly LeuPro Ala Ala Asp Arg Thr Tyr Cys Phe Asp Gly Phe                140                 145                 150 Ser Gly CysAsn Phe Pro Ala Glu Thr Gly Ile Ala Leu Tyr Ser                155                 160                 165 Leu His AspMet Ser Pro Ser Asp Val Ala Glu Ala Met Phe Arg                170                 175                 180 His Gly MetThr Arg Leu Tyr Ala Ala Leu His Leu Pro Pro Glu                185                 190                 195 Val Leu LeuPro Pro Gly Thr Tyr Arg Thr Ala Ser Tyr Leu Leu                200                 205                 210 Ile His AspGly Arg Arg Val Val Val Thr Tyr Glu Gly Asp Thr                215                 220                 225 Ser Ala GlyTyr Asn His Asp Val Ser Asn Leu Arg Ser Trp Ile                230                 235                 240 Arg Thr ThrLys Val Thr Gly Asp His Pro Leu Val Ile Glu Arg                245                 250                 255 Val Arg AlaIle Gly Cys His Phe Val Leu Leu Leu Thr Ala Ala                260                 265                 270 Pro Glu ProSer Pro Met Pro Tyr Val Pro Tyr Pro Arg Ser Thr                275                 280                 285 Glu Val TyrVal Arg Ser Ile Phe Gly Pro Gly Gly Thr Pro Ser                290                 295                 300 Leu Phe ProThr Ser Cys Ser Thr Lys Ser Thr Phe His Ala Val                305                 310                 315 Pro Ala HisIle Trp Asp Arg Leu Met Leu Phe Gly Ala Thr Leu                320                 325                 330 Asp Asp GlnAla Phe Cys Cys Ser Arg Leu Met Thr Tyr Leu Arg                335                 340                 345 Gly Ile SerTyr Lys Val Thr Val Gly Thr Leu Val Ala Asn Glu                350                 355                 360 Gly Trp AsnAla Ser Glu Asp Ala Leu Thr Ala Val Ile Thr Ala                365                 370                 375 Ala Tyr LeuThr Ile Cys His Gln Arg Tyr Leu Arg Thr Gln Ala                380                 385                 390 Ile Ser LysGly Met Arg Arg Leu Glu Arg Glu His Ala Gln Lys                395                 400                 405 Phe Ile ThrArg Leu Tyr Ser Trp Leu Phe Glu Lys Ser Gly Arg                410                 415                 420 Asp Tyr IlePro Gly Arg Gln Leu Glu Phe Tyr Ala Gln Cys Arg                425                 430                 435 Arg Trp LeuSer Ala Gly Phe His Leu Asp Pro Arg Val Leu Val                440                 445                 450 Phe Asp GluSer Ala Pro Cys His Cys Arg Thr Ala Ile Arg Lys                455                 460                 465 Ala Val SerLys Phe Cys Cys Phe Met Lys Trp Leu Gly Gln Glu                470                 475                 480 Cys Thr CysPhe Leu Gln Pro Ala Glu Gly Val Val Gly Asp Gln                485                 490                 495 Gly His AspAsn Glu Ala Tyr Glu Gly Ser Asp Val Asp Pro Ala                500                 505                 510 Glu Ser AlaIle Ser Asp Ile Ser Gly Ser Tyr Val Val Pro Gly                515                 520                 525 Thr Ala LeuGln Pro Leu Tyr Gln Ala Leu Asp Leu Pro Ala Glu                530                 535                 540 Ile Val AlaArg Ala Gly Arg Leu Thr Ala Thr Val Lys Val Ser                545                 550                 555 Gln Val AspGly Arg Ile Asp Cys Glu Thr Leu Leu Gly Asn Lys                560                 565                 570 Thr Phe ArgThr Ser Phe Val Asp Gly Ala Val Leu Glu Thr Asn                575                 580                 585 Gly Pro GluArg His Asn Leu Ser Phe Asp Ala Ser Gln Ser Thr                590                 595                 600 Met Ala AlaGly Pro Phe Ser Leu Thr Tyr Ala Ala Ser Ala Ala                605                 610                 615 Gly Leu GluVal Arg Tyr Val Ala Ala Gly Leu Asp His Arg Ala                620                 625                 630 Val Phe AlaPro Gly Val Ser Pro Arg Ser Ala Pro Gly Glu Val                635                 640                 645 Thr Ala PheCys Ser Ala Leu Tyr Arg Phe Asn Arg Glu Ala Gln                650                 655                 660 Arg Leu SerLeu Thr Gly Asn Phe Trp Phe His Pro Glu Gly Leu                665                 670                 675 Leu Gly ProPhe Ala Pro Phe Ser Pro Gly His Val Trp Glu Ser                680                 685                 690 Ala Asn ProPhe Cys Gly Glu Ser Thr Leu Tyr Thr Arg Thr Trp                695                 700                 705 Ser Glu ValAsp Ala Val Pro Ser Pro Ala Gln Pro Asp Leu Gly                710                 715                 720 Phe Thr SerGlu Pro Ser Ile Pro Ser Arg Ala Ala Thr Pro Thr                725                 730                 735 Pro Ala AlaPro Leu Pro Pro Pro Ala Pro Asp Pro Ser Pro Thr                740                 745                 750 Leu Ser AlaPro Ala Arg Gly Glu Pro Ala Pro Gly Ala Thr Ala                755                 760                 765 Arg Ala ProAla Ile Thr His Gln Thr Ala Arg His Arg Arg Leu                770                 775                 780 Leu Phe ThrTyr Pro Asp Gly Ser Lys Val Phe Ala Gly Ser Leu                785                 790                 795 Phe Glu SerThr Cys Thr Trp Leu Val Asn Ala Ser Asn Val Asp                800                 805                 810 His Arg ProGly Gly Gly Leu Cys His Ala Phe Tyr Gln Arg Tyr                815                 820                 825 Pro Ala SerPhe Asp Ala Ala Ser Phe Val Met Arg Asp Gly Ala                830                 835                 840 Ala Ala TyrThr Leu Thr Pro Arg Pro Ile Ile His Ala Val Ala                845                 850                 855 Pro Asp TyrArg Leu Glu His Asn Pro Lys Arg Leu Glu Ala Ala                860                 865                 870 Tyr Arg GluThr Cys Ser Arg Leu Gly Thr Ala Ala Tyr Pro Leu                875                 880                 885 Leu Gly ThrGly Ile Tyr Gln Val Pro Ile Gly Pro Ser Phe Asp                890                 895                 900 Ala Trp GluArg Asn His Arg Pro Gly Asp Glu Leu Tyr Leu Pro                905                 910                 915 Glu Leu AlaAla Arg Trp Phe Glu Ala Asn Arg Pro Thr Cys Pro                920                 925                 930 Thr Leu ThrIle Thr Glu Asp Val Ala Arg Thr Ala Asn Leu Ala                935                 940                 945 Ile Glu LeuAsp Ser Ala Thr Asp Val Gly Arg Ala Cys Ala Gly                950                 955                 960 Cys Arg ValThr Pro Gly Val Val Gln Tyr Gln Phe Thr Ala Gly                965                 970                 975 Val Pro GlySer Gly Lys Ser Arg Ser Ile Thr Gln Ala Asp Val                980                 985                 990 Asp Val ValVal Val Pro Thr Arg Glu Leu Arg Asn Ala Trp Arg                995                1000                1005 Arg Arg GlyPhe Ala Ala Phe Thr Pro His Thr Ala Ala Arg Val                1010               1015                1020 Thr Gln GlyArg Arg Val Val Ile Asp Glu Ala Pro Ser Leu Pro                1025               1030                1035 Pro His LeuLeu Leu Leu His Met Gln Arg Ala Ala Thr Val His                1040               1045                1050 Leu Leu GlyAsp Pro Asn Gln Ile Pro Ala Ile Asp Phe Glu His                1055               1060                1065 Ala Gly LeuVal Pro Ala Ile Arg Pro Asp Leu Ala Pro Thr Ser                1070               1075                1080 Trp Trp HisVal Thr His Arg Cys Pro Ala Asp Val Cys Glu Leu                1085               1090                1095 Ile Arg GlyAla Tyr Pro Met Ile Gln Thr Thr Ser Arg Val Leu                1100               1105                1110 Arg Ser LeuPhe Trp Gly Glu Pro Ala Val Gly Gln Lys Leu Val                1115               1120                1125 Phe Thr GlnAla Ala Lys Ala Ala Asn Pro Gly Ser Val Thr Val                1130               1135                1140 His Glu AlaGln Gly Ala Thr Tyr Thr Glu Thr Thr Ile Ile Ala                1145               1150                1155 Thr Ala AspAla Arg Gly Leu Ile Gln Ser Ser Arg Ala His Ala                1160               1165                1170 Ile Val AlaLeu Thr Arg His Thr Glu Lys Cys Val Ile Ile Asp                1175               1180                1185 Ala Pro GlyLeu Leu Arg Glu Val Gly Ile Ser Asp Ala Ile Val                1190               1195                1200 Asn Asn PhePhe Leu Ala Gly Gly Glu Ile Gly His Gln Arg Pro                1205               1210                1215 Ser Val IlePro Arg Gly Asn Pro Asp Ala Asn Val Asp Thr Leu                1220               1225                1230 Ala Ala PhePro Pro Ser Cys Glu Ile Ser Ala Phe His Glu Leu                1235               1240                1245 Ala Glu GluLeu Gly His Arg Pro Ala Pro Val Ala Ala Val Leu                1250               1255                1260 Pro Pro CysPro Glu Leu Glu Gln Gly Leu Leu Tyr Leu Pro Gln                1265               1270                1275 Glu Leu ThrThr Cys Asp Ser Val Val Thr Phe Glu Leu Thr Asp                1280               1285                1290 Ile Val HisCys Arg Met Ala Ala Pro Ser Gln Arg Lys Ala Val                1295               1300                1305 Leu Ser ThrLeu Val Gly Arg Tyr Gly Arg Arg Thr Lys Leu Tyr                1310               1315                1320 Asn Ala SerHis Ser Asp Val Arg Asp Ser Leu Ala Arg Phe Ile                1325               1330                1335 Pro Ala IleGly Pro Val Gln Val Thr Thr Cys Glu Leu Tyr Glu                1340               1345                1350 Leu Glu GluAla Met Val Glu Lys Gly Gln Asp Gly Ser Ala Val                1355               1360                1365 Leu Glu LeuAsp Leu Cys Ser Arg Asp Val Ser Arg Ile Thr Phe                1370               1375                1380 Phe Gln LysAsp Cys Asn Lys Phe Thr Thr Gly Glu Thr Ile Ala                1385               1390                1395 His Gly LysVal Gly Gln Gly Ile Ser Ala Trp Ser Lys Thr Phe                1400               1405                1410 Cys Ala LeuPhe Gly Pro Trp Phe Arg Ala Ile Glu Lys Ala Ile                1415               1420                1425 Leu Ala LeuLeu Pro Gln Gly Val Phe Tyr Gly Asp Ala Phe Asp                1430               1435                1440 Asp Thr ValPhe Ser Ala Ala Val Ala Ala Ala Lys Ala Ser Met                1445               1450                1455 Val Phe GluAsn Asp Phe Ser Glu Phe Asp Ser Thr Gln Asn Asn                1460               1465                1470 Phe Ser LeuGly Leu Glu Cys Ala Ile Met Glu Glu Cys Gly Met                1475               1480                1485 Pro Gln TrpLeu Ile Arg Leu Tyr His Leu Ile Arg Ser Ala Trp                1490               1495                1500 Ile Leu GlnAla Pro Lys Glu Ser Leu Arg Gly Phe Trp Lys Lys                1505               1510                1515 His Ser GlyGlu Pro Gly Thr Leu Leu Trp Asn Thr Val Trp Asn                1520               1525                1530 Met Ala ValIle Thr His Cys Tyr Asp Phe Arg Asp Leu Gln Val                1535               1540                1545 Ala Ala PheLys Gly Asp Asp Ser Ile Val Leu Cys Ser Glu Tyr                1550               1555                1560 Arg Gln SerPro Gly Ala Ala Val Leu Ile Ala Gly Cys Gly Leu                1565               1670                1575 Lys Leu LysVal Asp Phe Arg Pro Ile Gly Leu Tyr Ala Gly Val                1580               1585                1590 Val Val AlaPro Gly Leu Gly Ala Leu Pro Asp Val Val Arg Phe                1595               1600                1605 Ala Gly ArgLeu Thr Glu Lys Asn Trp Gly Pro Gly Pro Glu Arg                1610               1615                1620 Ala Glu GlnLeu Arg Leu Ala Val Ser Asp Phe Leu Arg Lys Leu                1625               1630                1635 Thr Asn ValAla Gln Met Cys Val Asp Val Val Ser Arg Val Tyr                1640               1645                1650 Gly Val SerPro Gly Leu Val His Asn Leu Ile Glu Met Leu Gln                1655               1660                1665 Ala Val AlaAsp Gly Lys Ala His Phe Thr Glu Ser Val Lys Pro                1670               1675                1680 Val Leu AspLeu Thr Asn Ser Ile Leu Cys Arg Val Glu                1685                1690

                     (SEQ. ID NO.: 2) Met Arg Pro Arg Pro Ile Leu LeuLeu Leu Leu Met Phe Leu Pro1               5                   10                   15 Met Leu ProAla Pro Pro Pro Gly Gln Pro Ser Gly Arg Arg Arg                20                  25                   30 Gly Arg ArgSer Gly Gly Ser Gly Gly Gly Phe Trp Gly Asp Arg                35                  40                   45 Val Asp SerGln Pro Phe Ala Ile Pro Tyr Ile His Pro Thr Asn                50                  55                   60 Pro Phe AlaPro Asp Val Thr Ala Ala Ala Gly Ala Gly Pro Arg                65                  70                   75 Val Arg GlnPro Ala Arg Pro Leu Gly Ser Ala Trp Arg Asp Gln                80                  85                   90 Ala Gln ArgPro Ala Ala Ala Ser Arg Arg Arg Pro Thr Thr Ala                95                  100                 105 Gly Ala AlaPro Leu Thr Ala Val Ala Pro Ala His Asp Thr Pro                110                 115                 120 Pro Val ProAsp Val Asp Ser Arg Gly Ala Ile Leu Arg Arg Gln                125                 130                 135 Tyr Asn LeuSer Thr Ser Pro Leu Thr Ser Ser Val Ala Thr Gly                140                 145                 150 Thr Asn LeuVal Leu Tyr Ala Ala Pro Leu Ser Pro Leu Leu Pro                155                 160                 165 Leu Gln AspGly Thr Asn Thr His Ile Met Ala Thr Glu Ala Ser                170                 175                 180 Asn Tyr AlaGln Tyr Arg Val Ala Arg Ala Thr Ile Arg Tyr Arg                185                 190                 195 Pro Leu ValPro Asn Ala Val Gly Gly Tyr Ala Ile Ser Ile Ser                200                 205                 210 Phe Tyr ProGln Thr Thr Thr Thr Pro Thr Ser Val Asp Met Asn                215                 220                 225 Ser Ile ThrSer Thr Asp Val Arg Ile Leu Val Gln Pro Gly Ile                230                 235                 240 Ala Ser GluLeu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn                245                 250                 255 Gln Gly TrpArg Ser Val Glu Thr Ser Gly Val Ala Glu Glu Glu                260                 265                 270 Ala Thr SerGly Leu Val Met Leu Cys Ile His Gly Ser Pro Val                275                 280                 285 Asn Ser TyrThr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu                290                 295                 300 Asp Phe AlaLeu Glu Leu Glu Phe Arg Asn Leu Thr Pro Gly Asn                305                 310                 315 Thr Asn ThrArg Val Ser Arg Tyr Ser Ser Thr Ala Arg His Arg                320                 325                 330 Leu Arg ArgGly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala                335                 340                 345 Ala Thr ArgPhe Met Lys Asp Leu Tyr Phe Thr Ser Thr Asn Gly                350                 355                 360 Val Gly GluIle Gly Arg Gly Ile Ala Leu Thr Leu Phe Asn Leu                365                 370                 375 Ala Asp ThrLeu Leu Gly Gly Leu Pro Thr Glu Leu Ile Ser Ser                380                 385                 390 Ala Gly GlyGln Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn                395                 400                 405 Gly Glu ProThr Val Lys Leu Tyr Thr Ser Val Glu Asn Ala Gln                410                 415                 420 Gln Asp LysGly Ile Ala Ile Pro His Asp Ile Asp Leu Gly Glu                425                 430                 435 Ser Arg ValVal Ile Gln Asp Tyr Asp Asn Gln His Glu Gln Asp                440                 445                 450 Arg Pro ThrPro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu                455                 460                 465 Arg Ala AsnAsp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr                470                 475                 480 Asp Gln SerThr Tyr Gly Ser Ser Thr Gly Pro Val Tyr Val Ser                485                 490                 495 Asp Ser ValThr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val                500                 505                 510 Ala Arg SerLeu Asp Trp Thr Lys Val Thr Leu Asp Gly Arg Pro                515                 520                 525 Leu Ser ThrIle Gln Gln Tyr Ser Lys Thr Phe Phe Val Leu Pro                530                 535                 540 Leu Arg GlyLys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala                545                 550                 555 Gly Tyr ProTyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Leu Leu                560                 565                 570 Val Glu AsnAla Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr                575                 580                 585 Thr Ser LeuGly Ala Gly Pro Val Ser Ile Ser Ala Val Ala Val                590                 595                 600 Leu Ala ProHis Ser Val Leu Ala Leu Leu Glu Asp Thr Met Asp                605                 610                 615 Tyr Pro AlaArg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys                620                 625                 630 Arg Pro LeuGly Leu Gln Gly Cys Ala Phe Gln Ser Thr Val Ala                635                 640                 645 Glu Leu GlnArg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Leu                650                 655                 660

                     (SEQ. ID NO.: 3) Met Asn Asn Met Ser Phe Ala AlaPro Met Gly Ser Arg Pro Cys1               5                    10                  15 Ala Leu GlyLeu Phe Cys Cys Cys Ser Ser Cys Phe Cys Leu Cys                20                   25                  30 Cys Pro ArgHis Arg Pro Val Ser Arq Leu Ala Ala Val Val Gly                35                   40                  45 Gly Ala AlaAla Val Pro Ala Val Val Ser Gly Val Thr Gly Leu                50                   55                  60 Ile Leu SerPro Ser Gln Ser Pro Ile Phe Ile Gln Pro Thr Pro                65                   70                  75 Ser Pro ProMet Ser Pro Leu Arg Pro Gly Leu Asp Leu Val Phe                80                   85                  90 Ala Asn ProPro Asp His Ser Ala Pro Leu Gly Val Thr Arg Pro                95                  100                 105 Ser Ala ProPro Leu Pro His Val Val Asp Leu Pro Gln Leu Gly                110                 115                 120 Pro Arg Arg

The three-letter abbreviations follow the conventional amino acidshorthand for the twenty naturally occuring amino acids.

The preferred recombinant HEV proteins consist of at least one ORFprotein. Other recombinant proteins made up of more than one of the sameor different ORF proteins may be made to alter the biological propertiesof the protein. It is contemplated that additions, substitutions ordeletions of discrete amino acids or of discrete sequences of aminoacids may enhance the biological activity of the HEV proteins.

The present invention is also a nucleic acid sequence which is capableof directing the production of the above-discussed HEV protein orproteins substantially homologous to the HEV proteins. This nucleic acidsequence, designated SAR-55, is set forth below as SEQ ID NO.: 4 and wasdeposited with the American Type Culture Collection (ATCC) on Sep. 17,1992 (ATCC accession number 75302).

AGGCAGACCA CATATGTGGT CGATGCCATG GAGGCCCATC 40 AGTTTATCAA GGCTCCTGGCATCACTACTG CTATTGAGCA 80 GGCTGCTCTA GCAGCGGCCA ACTCTGCCCT TGCGAATGCT 120GTGGTAGTTA GGCCTTTTCT CTCTCACCAG CAGATTGAGA 160 TCCTTATTAA CCTAATGCAACCTCGCCAGC TTGTTTTCCG 200 CCCCGAGGTT TTCTGGAACC ATCCCATCCA GCGTGTTATC240 CATAATGAGC TGGAGCTTTA CTGTCGCGCC CGCTCCGGCC 280 GCTGCCTCGAAATTGGTGCC CACCCCCGCT CAATAAATGA 320 CAATCCTAAT GTGGTCCACC GTTGCTTCCTCCGTCCTGCC 360 GGGCGTGATG TTCAGCGTTG GTATACTGCC CCTACCCGCG 400GGCCGGCTGC TAATTGCCGG CGTTCCGCGC TGCGCGGGCT 440 CCCCGCTGCT GACCGCACTTACTGCTTCGA CGGGTTTTCT 480 GGCTGTAACT TTCCCGCCGA GACGGGCATC GCCCTCTATT520 CTCTCCATGA TATGTCACCA TCTGATGTCG CCGAGGCTAT 560 GTTCCGCCATGGTATGACGC GGCTTTACGC TGCCCTCCAC 600 CTCCCGCCTG AGGTCCTGTT GCCCCCTGGCACATACCGCA 640 CCGCGTCGTA CTTGCTGATC CATGACGGCA GGCGCGTTGT 680GGTGACGTAT GAGGGTGACA CTAGTGCTGG TTATAACCAC 720 GATGTTTCCA ACCTGCGCTCCTGGATTAGA ACCACTAAGG 760 TTACCGGAGA CCACCCTCTC GTCATCGAGC GGGTTAGGGC800 CATTGGCTGC CACTTTGTCC TTTTACTCAC GGCTGCTCCG 840 GAGCCATCACCTATGCCCTA TGTCCCTTAC CCCCGGTCTA 880 CCGAGGTCTA TGTCCGATCG ATCTTCGGCCCGGGTGGCAC 920 CCCCTCCCTA TTTCCAACCT CATGCTCCAC CAAGTCGACC 960TTCCATGCTG TCCCTGCCCA TATCTGGGAC CGTCTCATGT 1000 TGTTCGGGGC CACCCTAGATGACCAAGCCT TTTGCTGCTC 1040 CCGCCTAATG ACTTACCTCC GCGGCATTAG CTACAAGGTT1080 ACTGTGGGCA CCCTTGTTGC CAATGAAGGC TGGAACGCCT 1120 CTGAGGACGCTCTTACAGCT GTCATCACTG CCGCCTACCT 1160 TACCATCTGC CACCAGCGGT ACCTCCGCACTCAGGCTATA 1200 TCTAAGGGGA TGCGTCGCCT GGAGCGGGAG CATGCTCAGA 1240AGTTTATAAC ACGCCTCTAC AGTTGGCTCT TTGAGAAGTC 1280 CGGCCGTGAT TATATCCCCGGCCGTCAGTT GGAGTTCTAC 1320 GCTCAGTGTA GGCGCTGGCT CTCGGCCGGC TTTCATCTTG1360 ACCCACGGGT GTGGGTTTTT GATGAGTCGG CCCCCTGCCA 1400 CTGTAGGACTGCGATTCGTA AGGCGGTCTC AAAGTTTTGC 1440 TGCTTTATGA AGTGGCTGGG CCAGGAGTGCACCTGTTTTC 1480 TACAACCTGC AGAAGGCGTC GTTGGCGACC AGGGCCATGA 1520CAACGAGGCC TATGAGGGGT CTGATGTTGA CCCTGCTGAA 1560 TCCGCTATTA GTGACATATCTGGGTCCTAC GTAGTCCCTG 1600 GCACTGCCCT CCAACCGCTT TACCAAGCCC TTGACCTCCC1640 CGCTGAGATT GTGGCTCGTG CAGGCCGGCT GACCGCCACA 1680 GTAAAGGTCTCCCAGGTCGA CGGGCGGATC GATTGTGAGA 1720 CCCTTCTCGG TAATAAAACC TTCCGCACGTCGTTTGTTGA 1760 CGGGGCGGTT TTAGAGACTA ATGGCCCAGA GCGCCACAAT 1800CTCTCTTTTG ATGCCAGTCA GAGCACTATG GCCGCCGGCC 1840 CTTTCAGTCT CACCTATGCCGCCTCTGCTG CTGGGCTGGA 1880 GGTGCGCTAT GTCGCCGCCG GGCTTGACCA CCGGGCGGTT1920 TTTGCCCCCG GCGTTTCACC CCGGTCAGCC CCTGGCGAGG 1960 TCACCGCCTTCTGTTCTGCC CTATACAGGT TTAATCGCGA 2000 GGCCCAGCGC CTTTCGCTGA CCGGTAATTTTTGGTTCCAT 2040 CCTGAGGGGC TCCTTGGCCC CTTTGCCCCG TTTTCCCCCG 2080GGCATGTTTG GGAGTCGGCT AATCCATTCT GTGGCGAGAG 2120 CACACTTTAC ACCCGCACTTGGTCGGAGGT TGATGCTGTT 2160 CCTAGTCCAG CCCAGCCCGA CTTAGGTTTT ACATCTGAGC2200 CTTCTATACC TAGTAGGGCC GCCACACCTA CCCCGGCGGC 2240 CCCTCTACCCCCCCCTGCAC CGGATCCTTC CCCTACTCTC 2280 TCTGCTCCGG CGCGTGGTGA GCCGGCTCCTGGCGCTACCG 2320 CCAGGGCCCC AGCCATAACC CACCAGACGG CCCGGCATCG 2360CCGCCTGCTC TTTACCTACC CGGATGGCTC TAAGGTGTTC 2400 GCCGGCTCGC TGTTTGAGTCGACATGTACC TGGCTCGTTA 2440 ACGCGTCTAA TGTTGACCAC CGCCCTGGCG GTGGGCTCTG2480 TCATGCATTT TACCAGAGGT ACCCCGCCTC CTTTGATGCT 2520 GCCTCTTTTGTGATGCGCGA CGGCGCGGCC GCCTACACAT 2560 TAACCCCCCG GCCAATAATT CATGCCGTCGCTCCTGATTA 2600 TAGGTTGGAA CATAACCCAA AGAGGCTTGA GGCTGCCTAC 2640CGGGAGACTT GCTCCCGCCT CGGTACCGCT GCATACCCAC 2680 TCCTCGGGAC CGGCATATACCAGGTGCCGA TCGGTCCCAG 2720 TTTTGACGCC TGGGAGCGGA ATCACCGCCC CGGGGACGAG2760 TTGTACCTTC CTGAGCTTGC TGCCAGATGG TTCGAGGCCA 2800 ATAGGCCGACCTGCCCAACT CTCACTATAA CTGAGGATGT 2840 TGCGCGGACA GCAAATCTGG CTATCGAACTTGACTCAGCC 2880 ACAGACGTCG GCCGGGCCTG TGCCGGCTGT CGAGTCACCC 2920CCGGCGTTGT GCAGTACCAG TTTACCGCAG GTGTGCCTGG 2960 ATCCGGCAAG TCCCGCTCTATTACCCAAGC CGACGTGGAC 3000 GTTGTCGTGG TCCCGACCCG GGAGTTGCGT AATGCCTGGC3040 GCCGCCGCGG CTTCGCTGCT TTCACCCCGC ACACTGCGGC 3080 TAGAGTCACCCAGGGGCGCC GGGTTGTCAT TGATGAGGCC 3120 CCGTCCCTTC CCCCTCATTT GCTGCTGCTCCACATGCAGC 3160 GGGCCGCCAC CGTCCACCTT CTTGGCGACC CGAATCAGAT 3200CCCAGCCATC GATTTTGAGC ACGCCGGGCT CGTTCCCGCC 3240 ATCAGGCCCG ATTTGGCCCCCACCTCCTGG TGGCATGTTA 3280 CCCATCGCTG CCCTGCGGAT GTATGTGAGC TAATCCGCGG3320 CGCATACCCT ATGATTCAGA CCACTAGTCG GGTCCTCCGG 3360 TCGTTGTTCTGGGGTGAGCC CGCCGTTGGG CAGAAGCTAG 3400 TGTTCACCCA GGCGGCTAAG GCCGCCAACCCCGGTTCAGT 3440 GACGGTCCAT GAGGCACAGG GCGCTACCTA CACAGAGACT 3480ACCATCATTG CCACGGCAGA TGCTCGAGGC CTCATTCAGT 3520 CGTCCCGAGC TCATGCCATTGTTGCCTTGA CGCGCCACAC 3560 TGAGAAGTGC GTCATCATTG ACGCACCAGG CCTGCTTCGC3600 GAGGTGGGCA TCTCCGATGC AATCGTTAAT AACTTTTTCC 3640 TTGCTGGTGGCGAAATTGGC CACCAGCGCC CATCTGTTAT 3680 CCCTCGCGGC AATCCTGACG CCAATGTTGACACCTTGGCT 3720 GCCTTCCCGC CGTCTTGCCA GATTAGCGCC TTCCATCAGT 3760TGGCTGAGGA GCTTGGCCAC AGACCTGCCC CTGTCGCGGC 3800 TGTTCTACCG CCCTGCCCTGAGCTTGAACA GGGCCTTCTC 3840 TACCTGCCCC AAGAACTCAC CACCTGTGAT AGTGTCGTAA3880 CATTTGAATT AACAGATATT GTGCATTGTC GTATGGCCGC 3920 CCCGAGCCAGCGCAAGGCCG TGCTGTCCAC GCTCGTGGGC 3960 CGTTATGGCC GCCGCACAAA GCTCTACAATGCCTCCCACT 4000 CTGATGTTCG CGACTCTCTC GCCCGTTTTA TCCCGGCCAT 4040TGGCCCCGTA CAGGTTACAA CCTGTGAATT GTACGAGCTA 4080 GTGGAGGCCA TGGTCGAGAAGGGCCAGGAC GGCTCCGCCG 4120 TCCTTGAGCT CGACCTTTGT AGCCGCGACG TGTCCAGGAT4160 CACCTTCTTC CAGAAAGATT GTAATAAATT CACCACGGGG 4200 GAGACCATCGCCCATGGTAA AGTGGGCCAG GGCATTTCGG 4240 CCTGGAGTAA GACCTTCTGT GCCCTTTTCGGCCCCTGGTT 4280 CCGTGCTATT GAGAAGGCTA TCCTGGCCCT GCTCCCTCAG 4320GGTGTGTTTT ATGGGGATGC CTTTGATGAC ACCGTCTTCT 4360 CGGCGGCTGT GGCCGCAGCAAAGGCATCCA GAATGACTTT 4400 TCTGAGTTTG ATTCCACCCA GAATAATTTT TCCTTGGGCC4440 TAGAGTGTGC TATTATGGAG GAGTGTGGGA TGCCGCAGTG 4480 GCTCATCCGCTTGTACCACC TTATAAGGTC TGCGTGGATT 4520 CTGCAGGCCC CGAAGGAGTC CCTGCGAGGGTTTTGGAAGA 4560 AACACTCCGG TGAGCCCGGC ACCCTTCTGT GGAATACTGT 4600CTGGAACATG GCCGTTATCA CCCACTGTTA TGATTTCCGC 4640 GATCTGCAGG TGGCTGCCTTTAAAGGTGAT GATTCGATAG 4680 TGCTTTGCAG TGAGTACCGT CAGAGCCCAG GGGCTGCTGT4720 CCTGATTGCT GGCTGTGGCC TAAAGTTGAA GGTGGATTTC 4760 CGTCCGATTGGTCTGTATGC AGGTGTTGTG GTGGCCCCCG 4800 GCCTTGGCGC GCTTCCTGAT GTCGTGCGCTTCGCCGGTCG 4840 GCTTACTGAG AAGAATTGGG GCCCTGGCCC CGAGCGGGCG 4880GAGCAGCTCC GCCTCGCTGT GAGTGATTTT CTCCGCAAGC 4920 TCACGAATGT AGCTCAGATGTGTGTGGATG TTGTCTCTCG 4960 TGTTTATGGG GTTTCCCCTG GGCTCGTTCA TAACCTGATT5000 GGCATGCTAC AGGCTGTTGC TGATGGCAAG GCTCATTTCA 5040 CTGAGTCAGTGAAGCCAGTG CTTGACCTGA CAAATTCAAT 5080 TCTGTGTCGG GTGGAATGAA TAACATGTCTTTTGCTGCGC 5120 CCATGGGTTC GCGACCATGC GCCCTCGGCC TATTTTGCTG 5160TTGCTCCTCA TGTTTCTGCC TATGCTGCCC GCGCCACCGC 5200 CCGGTCAGCC GTCTGGCCGCCGTCGTGGGC GGCGCAGCGG 5240 CGGTTCCGGC GGTGGTTTCT GGGGTGACCG GGTTGATTCT5280 CAGCCCTTCG CAATCCCCTA TATTCATCCA ACCAACCCCT 5320 TCGCCCCCGATGTCACCGCT GCGGCCGGGG CTGGACCTCG 5360 TGTTCGCCAA CCCGCCCGAC CACTCGGCTCCGCTTGGCGT 5400 GACCAGGCCC AGCGCCCCGC CGCTGCCTCA CGTCGTAGAC 5440CTACCACAGC TGGGGCCGCG CCGCTAACCG CGGTCGCTCC 5480 GGCCCATGAC ACCCCGCCAGTGCCTGATGT TGACTCCCGC 5520 GGCGCCATCC TGCGCCGGCA GTATAACCTA TCAACATCTC5560 CCCTCACCTC TTCCGTGGCC ACCGGCACAA ATTTGGTTCT 5600 TTACGCCGCTCCTCTTAGCC CGCTTCTACC CCTCCAGGAC 5640 GGCACCAATA CTCATATAAT GGCTACAGAAGCTTCTAATT 5680 ATGCCCAGTA CCGGGTTGCT CGTGCCACAA TTCGCTACCG 5720CCCGCTGGTC CCCAACGCTG TTGGTGGCTA CGCTATCTCC 5760 ATTTCGTTCT GGCCACAGACCACCACCACC CCGACGTCCG 5800 TTGACATGAA TTCAATAACC TCGACGGATG TCCGTATTTT5840 AGTCCAGCCC GGCATAGCCT CCGAGCTTGT TATTCCAAGT 5880 GAGCGCCTACACTATCGCAA CCAAGGTTGG CGCTCTGTTG 5920 AGACCTCCGG GGTGGCGGAG GAGGAGGCCACCTCTGGTCT 5960 TGTCATGCTC TGCATACATG GCTCACCTGT AAATTCTTAT 6000ACTAATACAC CCTATACCGG TGCCCTCGGG CTGTTGGACT 6040 TTGCCCTCGA ACTTGAGTTCCGCAACCTCA CCCCCGGTAA 6080 TACCAATACG CGGGTCTCGC GTTACTCCAG CACTGCCCGT6120 CACCGCCTTC GTCGCGGTGC AGATGGGACT GCCGAGCTCA 6160 CCACCACGGCTGCTACTCGC TTCATGAAGG ACCTCTATTT 6200 TACTAGTACT AATGGTGTTG GTGAGATCGGCCGCGGGATA 6240 GCGCTTACCC TGTTTAACCT TGCTGACACC CTGCTTGGCG 6280GTCTACCGAC AGAATTGATT TCGTCGGCTG GTGGCCAGCT 6320 GTTCTACTCT CGCCCCGTCGTCTCAGCCAA TGGCGAGCCG 6360 ACTGTTAAGC TGTATACATC TGTGGAGAAT GCTCAGCAGG6400 ATAAGGGTAT TGCAATCCCG CATGACATCG ACCTCGGGGA 6440 ATCCCGTGTAGTTATTCAGG ATTATGACAA CCAACATGAG 6480 CAGGACCGAC CGACACCTTC CCCAGCCCCATCGCGTCCTT 6520 TTTCTGTCCT CCGAGCTAAC GATGTGCTTT GGCTTTCTCT 6560CACCGCTGCC GAGTATGACC AGTCCACTTA CGGCTCTTCG 6600 ACCGGCCCAG TCTATGTCTCTGACTCTGTG ACCTTGGTTA 6640 ATGTTGCGAC CGGCGCGCAG GCCGTTGCCC GGTCACTCGA6680 CTGGACCAAG GTCACACTTG ATGGTCGCCC CCTTTCCACC 6720 ATCCAGCAGTATTCAAAGAC CTTCTTTGTC CTGCCGCTCC 6760 GCGGTAAGCT CTCCTTTTGG GAGGCAGGAACTACTAAAGC 6800 CGGGTACCCT TATAATTATA ACACCACTGC TAGTGACCAA 6840CTGCTCGTTG AGAATGCCGC TGGGCATCGG GTTGCTATTT 6880 CCACCTACAC TACTAGCCTGGGTGCTGGCC CCGTCTCTAT 6920 TTCCGCGGTT GCTGTTTTAG CCCCCCACTC TGTGCTAGCA6960 TTGCTTGAGG ATACCATGGA CTACCCTGCC CGCGCCCATA 7000 CTTTCGATGACTTCTGCCCG GAGTGCCGCC CCCTTGGCCT 7040 CCAGGGTTGT GCTTTTCAGT CTACTGTCGCTGAGCTTCAG 7080 CGCCTTAAGA TGAAGGTGGG TAAAACTCGG GAGTTATAGT 7120TTATTTGCTT GTGCCCCCCT TCTTTCTGTT GCTTATTT 7168

The abbreviations used for the nucleotides are those standardly used inthe art.

The sequence in one direction has been designated by convention as the“plus” sequence since it is the protein-encoding strand of RNA virusesand this is the sequence shown above as SEQ ID. NO.:4.

The deduced amino acid sequences of the open reading frames of SAR-55have SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3. ORF-1 starts atnucleotide 28 of SEQ. ID NO. 4 and extends 5078 nucleotides; ORF-2starts at nucleotide 5147 of SEQ. ID NO. 4 and extends 1979 nucleotides;and ORF-3 starts at nucleotide 5106 of SEQ. ID NO. 4 and extends 368nucleotides.

Variations are contemplated in the DNA sequence which will result in aDNA sequence that is capable of directing production of analogs of theORF-2 protein. By “analogs of the ORF-2 protein” as used throughout thespecification and claims is meant a protein having an amino acidsequence substantially identical to a sequence specifically shown hereinwhere one or more of the residues shown in the sequences presentedherein have been substituted with a biologically equivalent residue suchthat the resultant protein (i.e. the “analog”) is capable of formingviral particles and is immunogenic. It should be noted that the DNAsequence set forth above represents a preferred embodiment of thepresent invention. Due to the degeneracy of the genetic code, it is tobe understood that numerous choices of nucleotides may be made that willlead to a DNA sequence capable of directing production of the instantORF proteins or their analogs. As such, DNA sequences which arefunctionally equivalent to the sequences set forth above or which arefunctionally equivalent to sequences that would direct production ofanalogs of the ORF proteins produced pursuant to the amino acid sequenceset forth above, are intended to be encompassed within the presentinvention.

The present invention relates to a method for detecting the hepatitis Evirus in biological samples based on selective amplification ofhepatitis E gene fragments. Preferably, this method utilizes a pair ofsingle-stranded primers derived from non-homologous regions of oppositestrands of a DNA duplex fragment, which in turn is derived from ahepatitis E virus whose genome contains a region homologous to theSAR-55 sequence shown in SEQ ID No.: 4. These primers can be used in amethod following the process for amplifying selected nucleic acidsequences as defined in U.S. Pat. No. 4,683,202.

The present invention also relates to the use of single-strandedantisense poly-or oligonucleotides derived from sequences homologous tothe SAR-55 cDNA to inhibit the expression of hepatitis E genes. Theseanti-sense poly-or oligonucleotides can be either DNA or RNA. Thetargeted sequence is typically messenger RNA and more preferably, asignal sequence required for processing or translation of the RNA. Theantisense poly-or oligonucleotides can be conjugated to a polycationsuch as polylysine as disclosed in Lemaitre, M. et al. (1989) Proc NatlAcad Sci USA 84:648-652; and this conjugate can be administered to amammal in an amount sufficient to hybridize to and inhibit the functionof the messenger RNA.

The present invention includes a recombinant DNA method for themanufacture of HEV proteins, preferably a protein composed of at leastone ORF protein, most preferably at least one ORF-2 protein. Therecombinant ORF protein may be composed of one ORF protein or acombination of the same or different ORF proteins. A natural orsynthetic nucleic acid sequence may be used to direct production of theHEV proteins. In one embodiment of the invention, the method comprises:

(a) preparation of a nucleic acid sequence capable of directing a hostorganism to produce a protein of HEV;

(b) cloning the nucleic acid sequence into a vector capable of beingtransferred into and replicated in a host organism, such vectorcontaining operational elements for the nucleic acid sequence;

(c) transferring the vector containing the nucleic acid and operationalelements into a host organism capable of expressing the protein;

(d) culturing the host organism under conditions appropriate foramplification of the vector and expression of the protein; and

(e) harvesting the protein.

In another embodiment of the invention, the method for the recombinantDNA synthesis of a protein encoded by nucleic acids of HEV, preferablyencoding by at least one ORF of HEV or a combination of the same ordifferent ORF proteins, most preferably encoding at least one ORF-2nucleic acid sequence, comprises:

(a) culturing a transformed or transfected host organism containing anucleic acid sequence capable of directing the host organism to producea protein, under conditions such that the protein is produced, saidprotein exhibiting substantial homology to a native HEV protein isolatedfrom HEV having the amino acid sequence according to SEQ ID NO. 1, SEQID NO. 2 or SEQ ID NO. 3, or combinations thereof.

In one embodiment, the RNA sequence of the viral genome of HEV strainSAR-55 was isolated and cloned to cDNA as follows. Viral RNA isextracted from a biological sample collected from cynomolgus monkeysinfected with SAR-55 and the viral RNA is then reverse transcribed andamplified by polymerase chain reaction using primers complementary tothe plus or minus strands of the genome of a strain of HEV from Burma(Tam et al. (1991)) or the SAR-55 genome. The PCR fragments aresubcloned into pBR322 or pGEM-32 and the double-stranded PCR fragmentswere sequenced.

The vectors contemplated for use in the present invention include anyvectors into which a nucleic acid sequence as described above can beinserted, along with any preferred or required operational elements, andwhich vector can then be subsequently transferred into a host organismand replicated in such organism. Preferred vectors are those whoserestriction sites have been well documented and which contain theoperational elements preferred or required for transcription of thenucleic acid sequence.

The “operational elements” as discussed herein include at least onepromoter, at least one operator, at least one leader sequence, at leastone terminator codon, and any other DNA sequences necessary or preferredfor appropriate transcription and subsequent translation of the vectornucleic acid. In particular, it is contemplated that such vectors willcontain at least one origin of replication recognized by the hostorganism along with at least one selectable marker and at least onepromoter sequence capable of initiating transcription of the nucleicacid sequence.

In construction of the cloning vector of the present invention, itshould additionally be noted that multiple copies of the nucleic acidsequence and its attendant operational elements may be inserted intoeach vector. In such an embodiment, the host organism would producegreater amounts per vector of the desired HEV protein. The number ofmultiple copies of the DNA sequence which may be inserted into thevector is limited only by the ability of the resultant vector due to itssize, to be transferred into and replicated and transcribed in anappropriate host microorganism.

In another embodiment, restriction digest fragments containing a codingsequence for HEV proteins can be inserted into a suitable expressionvector that functions in prokaryotic or eukaryotic cells. By suitable ismeant that the vector is capable of carrying and expressing a completenucleic acid sequence coding for HEV proteins, preferably at least onecomplete ORF protein. In the case or ORF-2, the expressed protein shouldform viral-like particles. Preferred expression vectors are those thatfunction in a eukaryotic cell. Examples of such vectors include but arenot limited to vectors useful for expression in yeast (e.g. pPIC9vector-Invitrogen) vaccinia virus vectors, adenovirus or herpesviruses,preferably the baculovirus transfer vector, pBlueBac. Preferred vectorsare p63-2, which contains the complete ORF-2 gene, and P59-4, whichcontains the complete ORF-3 and ORF-2 genes. These vectors weredeposited with the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. 20852 USA on Sep. 10, 1992 and have accessionnumbers 75299 (P63-2) and 75300 (P59-4). Example 1 illustrates thecloning of the ORF-2 gene into pBlueBac to produce p63-2. This methodincludes digesting the genome of HEV strain SAR-55 with the restrictionenzymes NruI and BglII, inserting a polylinker containing BlnI and BglIIsites into the unique NheI site of the vector and inserting theNruI-BglII ORF-2 fragment in BlnI-BglII PBlueBac using an adapter.

In yet another embodiment, the selected recombinant expression vectormay then be transfected into a suitable eukaryotic cell system forpurposes of expressing the recombinant protein. Such eukaryotic cellsystems include, but are not limited to, yeast, and cell lines such asHeLa, MRC-5 or Cv-1. One preferred eukaryotic cell system is SF9 insectcells. One preferred method involves use of the pBlueBac expressionvector where the insect cell line SF9 is cotransfected with recombinantpBlueBac and AcMNPV baculovirus DNA by the Ca precipitation method.

The expressed recombinant protein may be detected by methods known inthe art which include Coomassie blue staining and Western blotting usingsera containing anti-HEV antibody as shown in Example 2. Another methodis the detection of virus-like particles by immunoelectron microscopy asshown in Example 3.

In a further embodiment, the recombinant protein expressed by the SF9cells can be obtained as a crude lysate or it can be purified bystandard protein purification procedures known in the art which mayinclude differential precipitation, molecular sieve chromatography,ion-exchange chromatography, isoelectric focusing, gel electrophoresis,affinity, and immunoaffinity chromatography and the like. In the case ofimmunoaffinity chromatography, the recombinant protein may be purifiedby passage through a column containing a resin which has bound theretoantibodies specific for the ORF protein. An example of a protocol forthe purification of a recombinantly expressed HEV ORF protein isprovided in Example 10.

In another embodiment, the expressed recombinant proteins of thisinvention can be used in immunoassays for diagnosing or prognosinghepatitis E in a mammal including but not limited to humans,chimpanzees, Old World monkeys, New World monkeys, other primates andthe like. In a preferred embodiment, the immunoassay is useful indiagnosing hepatitis E infection in humans. Immunoassays using the HEVproteins, particularly the ORF proteins, and especially ORF 2 proteins,provide a highly specific, sensitive and reproducible method fordiagnosing HEV infections, in contrast to immunoassays which utilizepartial ORF proteins. Immunoassays of the present invention may be aradioimmunoassay, Western blot assay, immunofluorescent assay, enzymeimmunoassay, chemiluminescent assay, immunohistochemical assay and thelike. Standard techniques known in the art for ELISA are described inMethods in Immunodiagnosis, 2nd Edition, Rose and Bigazzi, eds., JohnWiley and Sons, 1980 and Campbell et al., Methods of Immunology, W. A.Benjamin, Inc., 1964, both of which are incorporated herein byreference. Such assays may be a direct, indirect, competitive, ornoncompetitive immunoassay as described in the art. (Oellerich, M. 1984.J. Clin. Chem. Clin. BioChem. 22: 895-904) Biological samplesappropriate for such detection assays include, but are not limited to,tissue biopsy extracts, whole blood, plasma, serum, cerebrospinal fluid,pleural fluid, urine and the like.

In one embodiment, test serum is reacted with a solid phase reagenthaving surface-bound recombinant HEV protein as an antigen, preferablyan ORF protein or combination of different ORF proteins such as ORF-2and ORF-3, ORF-1 and ORF-3 and the like. More preferably, the HEVprotein is an ORF-2 protein that forms virus-like particles. The solidsurface reagent can be prepared by known techniques for attachingprotein to solid support material. These attachment methods includenon-specific adsorption of the protein to the support or covalentattachment of the protein to a reactive group on the support. Afterreaction of the antigen with anti-HEV antibody, unbound serum componentsare removed by washing and the antigen-antibody complex is reacted witha secondary antibody such as labelled anti-human antibody. The label maybe an enzyme which is detected by incubating the solid support in thepresence of a suitable fluorimetric or colorimetric reagent. Otherdetectable labels may also be used, such as radiolabels or colloidalgold, and the like.

In a preferred embodiment, protein expressed by the recombinant vectorpBlueBac containing the entire ORF-2 sequence of SAR-55 is used as aspecific binding agent to detect anti-HEV antibodies, preferably IgG orIgM antibodies. Examples 4 and 5 show the results of an ELISA in whichthe solid phase reagent has recombinant ORF-2 as the surface antigen.This protein, encoded by the entire ORF-2 nucleic acid sequence, issuperior to the partial ORF-2 proteins, as it is reactive with moreantisera from different primate species infected with HEV than arepartial antigens of ORF-2. The protein of the present invention is alsocapable of detecting antibodies produced in response to differentstrains of HEV but does not detect antibodies produced in response toHepatitis A, B, C or Hepatitis D.

The HEV protein and analogs may be prepared in the form of a kit, alone,or in combinations with other reagents such as secondary antibodies, foruse in immunoassays.

The recombinant HEV proteins, preferably an ORF protein or combinationof ORF proteins, more preferably an ORF-2 protein and substantiallyhomologous proteins and analogs of the invention can be used as avaccine to protect mammals against challenge with Hepatitis E. Thevaccine, which acts as an immunogen, may be a cell, cell lysate fromcells transfected with a recombinant expression vector or a culturesupernatant containing the expressed protein. Alternatively, theimmunogen is a partially or substantially purified recombinant protein.While it is possible for the immunogen to be administered in a pure orsubstantially pure form, it is preferable to present it as apharmaceutical composition, formulation or preparation.

The formulations of the present invention, both for veterinary and forhuman use, comprise an immunogen as described above, together with oneor more pharmaceutically acceptable carriers and optionally othertherapeutic ingredients. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethod well-known in the pharmaceutical art.

All methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired formulation.

Formulations suitable for intravenous intramuscular, subcutaneous, orintraperitoneal administration conveniently comprise sterile aqueoussolutions of the active ingredient with solutions which are preferablyisotonic with the blood of the recipient. Such formulations may beconveniently prepared by dissolving solid active ingredient in watercontaining physiologically compatible substances such as sodium chloride(e.g. 0.1-2.0M), glycine, and the like, and having a buffered pHcompatible with physiological conditions to produce an aqueous solution,and rendering said solution sterile. These may be present in unit ormulti-dose containers, for example, sealed ampoules or vials.

The formulations of the present invention may incorporate a stabilizer.Illustrative stabilizers are polyethylene glycol, proteins, saccharides,amino acids, inorganic acids, and organic acids which may be used eitheron their own or as admixtures. These stabilizers are preferablyincorporated in an amount of 0.11-10,000 parts by weight per part byweight of immunogen. If two or more stabilizers are to be used, theirtotal amount is preferably within the range specified above. Thesestabilizers are used in aqueous solutions at the appropriateconcentration and pH. The specific osmotic pressure of such aqueoussolutions is generally in the range of 0.1-3.0 osmoles, preferably inthe range of 0.8-1.2. The pH of the aqueous solution is adjusted to bewithin the range of 5.0-9.0, preferably within the range of 6-8. Informulating the immunogen of the present invention, anti-adsorptionagent may be used.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achievedthrough the use of polymer to complex or absorb the proteins or theirderivatives. The controlled delivery may be exercised by selectingappropriate macromolecules (for example polyester, polyamino acids,polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled-release preparations is to incorporate theproteins, protein analogs or their functional derivatives, intoparticles of a polymeric material such as polyesters, polyamino acids,hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these agents into polymericparticles, it is possible to entrap these materials in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxy-methylcellulose orgelatin-microcapsules and poly(methylmethacylate) microcapsules,respectively, or in colloidal drug delivery systems, for example,liposomes, albumin microspheres, microemulsions, nanoparticles, andnanocapsules or in macroemulsions.

When oral preparations are desired, the compositions may be combinedwith typical carriers, such as lactose, sucrose, starch, talc magnesiumstearate, crystalline cellulose, methyl cellulose, carboxymethylcellulose, glycerin, sodium alginate or gum arabic among others.

The proteins of the present invention may be supplied in the form of akit, alone, or in the form of a pharmaceutical composition as describedabove.

Vaccination can be conducted by conventional methods. For example, theimmunogen can be used in a suitable diluent such as saline or water, orcomplete or incomplete adjuvants. Further, the immunogen may or may notbe bound to a carrier to make the protein immunogenic. Examples of suchcarrier molecules include but are not limited to bovine serum albumin(BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid, and the like.The immunogen can be administered by any route appropriate for antibodyproduction such as intravenous, intraperitoneal, intramuscular,subcutaneous, and the like. The immunogen may be administered once or atperiodic intervals until a significant titer of anti-HEV antibody isproduced. The antibody may be detected in the serum using animmunoassay.

In yet another embodiment, the immunogen may be nucleic acid sequencecapable of directing host organism synthesis of an HEV ORF protein. Suchnucleic acid sequence may be inserted into a suitable expression vectorby methods known to those skilled in the art. Expression vectorssuitable for producing high efficiency gene transfer in vivo include,but are not limited to, retroviral, adenoviral and vaccinia viralvectors. Operational elements of such expression vectors are disclosedpreviously in the present specification and are known to one skilled inthe art. Such expression vectors can be administered intravenously,intramuscularly, subcutaneously, intraperitoneally or orally.

In an alternative embodiment, direct gene transfer may be accomplishedvia intramuscular injection of, for example, plasmid-based eukaryoticexpression vectors containing a nucleic acid sequence capable ofdirecting host organism synthesis HEC ORF protein(s). Such an approachhas previously been utilized to produce the hepatitis B surface antigenin vivo and resulted in an antibody response to the surface antigen(Davis, H. L. et al. (1993) Human Molecular Genetics, 2:1847-1851; seealso Davis et al. (1993) Human Gene Therapy, 4:151-159 and 733-740).

When the immunogen is a partially or substantially purified recombinantHEV ORF protein, dosages effective to elicit a protective antibodyresponse against HEV range from about 2 μg to about 100 μg. A morepreferred range is from about 5 μg to about 70 μg and a most preferredrange is from about 10 μg to about 60 μg.

Dosages of HEV-ORF protein-encoding nucleic acid sequence effective toelicit a protective antibody response against HEV range from about 1 toabout 5000 μg; a more preferred range being about 300 to about 1000 μg.

The expression vectors containing a nucleic acid sequence capable ofdirecting host organism synthesis of an HEV ORF protein(s) may besupplied in the form of a kit, alone, or in the form of a pharmaceuticalcomposition as described above.

The administration of the immunogen of the present invention may be foreither a prophylactic or therapeutic purpose. When providedprophylactically, the immunogen is provided in advance of any exposureto HEV or in advance of any symptom due to HEV infection. Theprophylactic administration of the immunogen serves to prevent orattenuate any subsequent infection of HEV in a mammal. When providedtherapeutically, the immunogen is provided at (or shortly after) theonset of the infection or at the onset of any symptom of infection ordisease caused by HEV. The therapeutic administration of the immunogenserves to attenuate the infection or disease.

A preferred embodiment is a vaccine prepared using recombinant ORF-2protein expressed by the ORF-2 sequence of HEV strain SAR-55 andequivalents thereof. Since the recombinant ORF-2 protein has alreadybeen demonstrated to be reactive with a variety of HEV-positive sera,their utility in protecting against a variety of HEV strains isindicated.

In addition to use as a vaccine, the compositions can be used to prepareantibodies to HEV virus-like particles. The antibodies can be useddirectly as antiviral agents. To prepare antibodies, a host animal isimmunized using the virus particles or, as appropriate, non-particleantigens native to the virus particle are bound to a carrier asdescribed above for vaccines. The host serum or plasma is collectedfollowing an appropriate time interval to provide a compositioncomprising antibodies reactive with the virus particle. The gammaglobulin fraction or the IgG antibodies can be obtained, for example, byuse of saturated ammonium sulfate or DEAE Sephadex, or other techniquesknown to those skilled in the art. The antibodies are substantially freeof many of the adverse side effects which may be associated with otheranti-viral agents such as drugs.

The antibody compositions can be made even more compatible with the hostsystem by minimizing potential adverse immune system responses. This isaccomplished by removing all or a portion of the Fc portion of a foreignspecies antibody or using an antibody of the same species as the hostanimal, for example, the use of antibodies from human/human hybridomas.Humanized antibodies (i.e., nonimmunogenic in a human) may be produced,for example, by replacing an immunogenic portion of an antibody with acorresponding, but nonimmunogenic portion (i.e., chimeric antibodies).Such chimeric antibodies may contain the reactive or antigen bindingportion of an antibody from one species and the Fc portion of anantibody (nonimmunogenic) from a different species. Examples of chimericantibodies, include but are not limited to, non-human mammal-humanchimeras, rodent-human chimeras, murine-human and rat-human chimeras(Robinson et al., International Patent Application 184,187; TaniguchiM., European Patent Application 171,496; Morrison et al., EuropeanPatent Application 173,494; Neuberger et al., PCT Application WO86/01533; Cabilly et al., 1987 Proc. Natl. Acad. Sci. USA 84:3439;Nishimura et al., 1987 Canc. Res. 47:999; Wood et al., 1985 Nature314:446; Shaw et al., 1988 J. Natl. Cancer Inst. 80: 15553, allincorporated herein by reference).

General reviews of “humanized” chimeric antibodies are provided byMorrison S., 1985 Science 229:1202 and by Oi et al., 1986 BioTechniques4:214.

Suitable “humanized” antibodies can be alternatively produced by CDR orCEA substitution (Jones et al., 1986 Nature 321:552; Verhoeyan et al.,1988 Science 239:1534; Biedleret al. 1988 J. Immunol. 141:4053, allincorporated herein by reference).

The antibodies or antigen binding fragments may also be produced bygenetic engineering. The technology for expression of both heavy andlight cain genes in E. coli is the subject the PCT patent applications;publication number WO 901443, WO901443, and WO 9014424 and in Huse etal., 1989 Science 246:1275-1281.

The antibodies can also be used as a means of enhancing the immuneresponse. The antibodies can be administered in amounts similar to thoseused for other therapeutic administrations of antibody. For example,pooled gamma globulin is administered at 0.02-0.1 ml/lb body weightduring the early incubation period of other viral diseases such asrabies, measles and hepatitis B to interfere with viral entry intocells. Thus, antibodies reactive with the HEV virus particle can bepassively administered alone or in conjunction with another anti-viralagent to a host infected with an HEV to enhance the effectiveness of anantiviral drug.

Alternatively, anti-HEV antibodies can be induced by administeringanti-idiotype antibodies as immunogens. Conveniently, a purifiedanti-HEV antibody preparation prepared as described above is used toinduce anti-idiotype antibody in a host animal. The composition isadministered to the host animal in a suitable diluent. Followingadministration, usually repeated administration, the host producesanti-idiotype antibody. To eliminate an immunogenic response to the Fcregion, antibodies produced by the same species as the host animal canbe used or the FC region of the administered antibodies can be removed.Following induction of anti-idiotype antibody in the host animal, serumor plasma is removed to provide an antibody composition. The compositioncan be purified as described above for anti-HEV antibodies, or byaffinity chromatography using anti-HEV antibodies bound to the affinitymatrix. The anti-idiotype antibodies produced are similar inconformation to the authentic HEV-antigen and may be used to prepare anHEV vaccine rather than using an HEV particle antigen.

When used as a means of inducing anti-HEV virus antibodies in an animal,the manner of injecting the anti-body is the same as for vaccinationpurposes, namely intramuscularly, intraperitoneally, subcutaneously orthe like in an effective concentration in a physiologically suitablediluent with or without adjuvant. One or more booster injections may bedesirable.

The HEV derived proteins of the invention are also intended for use inproducing antiserum designed for pre- or post-exposure prophylaxis. Herean HEV protein, or mixture of proteins is formulated with a suitableadjuvant and administered by injection to human volunteers, according toknown methods for producing human antisera. Antibody response to theinjected proteins is monitored, during a several-week period followingimmunization, by periodic serum sampling to detect the presence ofanti-HEV serum antibodies, using an immunoassay as described herein.

The antiserum from immunized individuals may be administered as apre-exposure prophylactic measure for individuals who are at risk ofcontracting infection. The antiserum is also useful in treating anindividual post-exposure, analogous to the use of high titer antiserumagainst hepatitis B virus for post-exposure prophylaxis. Of course,those of skill in the art would readily understand that immune globulin(HEV immune globulin) purified from the antiserum of immunizedindividuals using standard techniques may be used as a pre-exposureprophylactic measure or in treating individuals post-exposure.

For both in vivo use of antibodies to HEV virus-like particles andproteins and anti-idiotype antibodies and diagnostic use, it may bepreferable to use monoclonal antibodies. Monoclonal anti-virus particleantibodies or anti-idiotype antibodies can be produced as follows. Thespleen or lymphocytes from an immunized animal are removed andimmortalized or used to prepare hybridomas by methods known to thoseskilled in the art. (Goding, J. W. 1983. Monoclonal Antibodies:Principles and Practice, Pladermic Press, Inc., NY, N.Y., pp. 56-97). Toproduce a human-human hybridoma, a human lymphocyte donor is selected. Adonor known to be infected with HEV (where infection has been shown forexample by the presence of anti-virus antibodies in the blood or byvirus culture) may serve as a suitable lymphocyte donor. Lymphocytes canbe isolated from a peripheral blood sample or spleen cells may be usedif the donor is subject to splenectomy. Epstein-Barr virus (EBV) can beused to immortalize human lymphocytes or a human fusion partner can beused to produce human-human hybridomas. Primary in vitro immunizationwith peptides can also be used in the generation of human monoclonalantibodies.

Antibodies secreted by the immortalized cells are screened to determinethe clones that secrete antibodies of the desired specificity. Formonoclonal anti-virus particle antibodies, the antibodies must bind toHEV virus particles. For monoclonal anti-idiotype antibodies, theantibodies must bind to anti-virus particle antibodies. Cells producingantibodies of the desired specificity are selected.

The above described antibodies and antigen binding fragments thereof maybe supplied in kit form alone, or as a pharmaceutical composition for invivo use. The antibodies may be used for therapeutic uses, diagnosticuse in immunoassays or as an immunoaffinity agent to purify ORF proteinsas described herein.

Material

The materials used in the Examples were as follows:

Primates. Chimpanzee (Chimp) (Pan troglodytes). Old world monkeys:cynomolgus monkeys (Cyno) (Macaca fascicularis), rhesus monkeys (Rhesus)(M. mulatta), pigtail monkeys (PT) (M. nemestrina), and African greenmonkeys (AGM) (Cercopithecus aethiops). New World monkeys: mustachedtamarins (Tam) (Saguinus mystax), squirrel monkeys (SQM) (Saimirisciureus) and owl monkeys (OWL) (Aotus trivigatus). Primates were housedsingly under conditions of biohazard containment. The housing,maintenance and care of the animals met or exceeded all requirements forprimate husbandry.

Most animals were inoculated intravenously with HEV, strain SAR-55contained in 0.5 ml of stool suspension diluted in fetal calf serum asdescribed in Tsarev, S. A. et al. (1992), Proc. Natl. Acad. Sci USA,89:559-563; and Tsarev, S. A. et al. (1993), J. Infect. Dis.(167:1302-1306). Chimp-1313 and 1310 were inoculated with a pool ofstools collected from 7 Pakistani hepatitis E patients.

Serum samples were collected approximately twice a week before and afterinoculation. Levels of the liver enzymes serum alanine amino transferase(ALT), isocitrate dehydrogenase (ICD), and gamma glutamyl transferase(GGT) were assayed with commercially available tests (Medpath Inc.,Rockville, Md.). Serologic tests were performed as described above.

EXAMPLE 1 Identification of the DNA Sequence of the Genome of HEV StrainSAR-55

Preparation of Virus RNA Template for PCR. Bile from an HEV-infectedcynomolgus monkey (10 μl), 20% (wt/vol) SDS (to a final concentration of1%), proteinase K (10 mg/ml; to a final concentration of 1 mg/ml), 1 μlof tRNA (10 mg/ml), and 3 μl of 0.5 M EDTA were mixed in a final volumeof 250 μl and incubated for 30 min. at 55° C. Total nucleic acids wereextracted from bile twice with phenol/chloroform, 1:1 (vol/vol), at 65°C. and once with chloroform, then precipitated by ethanol, washed with95% ethanol, and used for RT-PCR. RT-PCR amplification of HEV RNA fromfeces and especially from sera was more efficient when RNA was moreextensively purified. Serum (100 μl) or a 10% fecal suspension (200 μl)was treated as above with proteinase K. After a 30-min incubation, 300μl of CHAOS buffer (4.2 M guanidine thiocyanate/0.5N-lauroylsarocosine/0.025 M Tris-HCL, pH 8.0) was added. Nucleic acidswere extracted twice with phenol/chloroform at 65° C. followed bychloroform extraction at room temperature. Then 7.5 M ammonium acetate(225 μl) was added to the upper phase and nucleic acids wereprecipitated with 0.68 ml of 2-propanol. The pellet was dissolved in 300ul CHAOS buffer and 100 ul of H₂O was added. Chloroform extraction and2-propanol precipitation were repeated. Nucleic acids were dissolved inwater, precipitated with ethanol, washed with 95% ethanol, and used forRT-PCR.

Primers. Ninety-four primers, 21-40 nucleotides (nt) long, andcomplementary to plus or minus strands of the genome of a strain of HEVfrom Burma (BUR-121) (Tam, A. W. et al. (1991), Virology, 185:120-131)or the SAR-55 genome were synthesized using an Applied Biosystems model391 DNA synthesizer.

The sequences of these 94 primers are shown below starting with SEQ. IDNO. 5 and continuing to SEQ. ID NO. 98:

HEV Primer List ORF Primer Region Sequence D 3042 B 1ACATTTGAATTCACAGACAT (SEQ. ID. NO. TGTGC 5) R 3043 B 1ACACAGATCTGAGCTACATT (SEQ. ID. NO. CGTGAG 6) D 3044 B 1AAAGGGATCCATGGTGTTTG (SEQ. ID. NO. AGAATGZ 7) R 3045 B 1ACTCACTGCAGAGCACTATC (SEQ. ID. NO. GAATC 8) R 261 S 1CGGTAAACTGGTACTGCACA (SEQ. ID. NO. AC 9) D 260 S 1 AAGTCCCGCTCTATTACCCA(SEQ. ID. NO. AG 10) D 259 S 1 ACCCACGGGTGTTGGTTTTT (SEQ. ID. NO. G 11)R 255 S 1 TTCTTGGGGCAGGTAGAGAA (SEQ. ID. NO. G 12) R 254 S 2TTATTGAATTCATGTCAACG (SEQ. ID. NO. GACGTC 13) D 242 S 1AATAATTCATGCCGTCGCTC (SEQ. ID. NO. C 14) R 241 S 1 AAGCTCAGGAAGGTACAACT(SEQ. ID. NO. C 15) R 231 S 1 AAATCGATGGCTGGGATCTG (SEQ. ID. NO. ATTC16) R 230 S 1 GAGGCATTGTAGAGCTTTGT (SEQ. ID. NO. G 17) D 229 S 1GATGTTGCACGGACAGCAAA (SEQ. ID. NO. TC 18) D 228 S 1 ATCTCCGATGCAATCGTTAA(SEQ. ID. NO. TAAC 19) D 227 B 1 TAATCCATTCTGTGGCGAGA (SEQ. ID. NO. G20) R 218 B 2 AAGTGTGACCTTGGTCCAGT (SEQ. ID. NO. C 21) D 217 B 2TTGCTCGTGCCACAATTCGC (SEQ. ID. NO. TAC 22) D 211 B 1CATTTCACTGAGTCAGTGAA (SEQ. ID. NO. G 23) D 202 B 2 TAATTATAACACCACTGCTA(SEQ. ID. NO. G 24) R 201 B 2 GATTGCAATACCCTTATCCT (SEQ. ID. NO. G 25) R200 S 1 ATTAAACCTGTATAGGGCAG (SEQ. ID. NO. AAC 26) R 199 S 1AAGTTCGATAGCCAGATTTG (SEQ. ID. NO. C 27) R 198 S 2 TCATGTTGGTTGTCATAATC(SEQ. ID. NO. C 28) R 193 B 1 GATGACGCACTTCTCAGTGT (SEQ. ID. NO. G 29) R192 B 1 AGAACAACGAACGGAGAAC (SEQ. ID. NO. 30) D 191 B 1AGATCCCAGCCATCGACTTT (SEQ. ID. NO. G 31) R 190 S 2 TAGTAGTGTAGGTGGAAATA(SEQ. ID. NO. G 32) D 189 B 2 GTGTGGTTATTCAGGATTAT (SEQ. ID. NO. G 33) D188 B 2 ACTCTGTGACCTTGGTTAAT (SEQ. ID. NO. G 34) R 187 S 2AACTCAAGTTCGAGGGCAAA (SEQ. ID. NO. G 35) D 186 S 2 CGCTTACCCTGTTTAACCTT(SEQ. ID. NO. G 36) D 185 B 2,3 ATCCCCTATATTCATCCAAC (SEQ. ID. NO. CAAC37) D 184 S 2,3 CTCCTCATGTTTCTGCCTAT (SEQ. ID. NO. G 38) R 181 S 2GCCAGAACGAAATGGAGATA (SEQ. ID. NO. GC 39) R 180 B 1 CTCAGACATAAAACCTAAGT(SEQ. ID. NO. C 40) D 179 S 1 TGCCCTATACAGGTTTAATC (SEQ. ID. NO. G 41) D178 B 1 ACCGGCATATACCAGGTGC (SEQ. ID. NO. 42) D 177 B 2ACATGGCTCACTCGTAAATT (SEQ. ID. NO. C 43) R 174 B 1 AACATTAGACGCGTTAACGA(SEQ. ID. NO. G 44) D 173 S 1 CTCTTTTGATGCCAGTCAGA (SEQ. ID. NO. G 45) D172 B 1 ACCTACCCGGATGGCTCTAA (SEQ. ID. NO. GG 46) R 166 B 2TATGGGAATTCGTGCCGTCC (SEQ. ID. NO. TGAAG (EcoRI) 47) R 143 B 1AGTGGGAGCAGTATACCAGC (SEQ. ID. NO. G 48) D 141 B 1 CTGCTATTGAGCAGGCTGCT(SEQ. ID. NO. C 49) R 142 S 1 GGGCCATTAGTCTCTAAAAC (SEQ. ID. NO. C 50) D135 B 1 GAGGTTTTCTGGAATCATC (SEQ. ID. NO. 51) R 134 B 1 GCATAGGTGAGACTG(SEQ. ID. NO. 52) R 133 B 1 AGTTACAGCCAGAAAACC (SEQ. ID. NO. 53) D 132 S2,3 CCATGGATCCTCGGCCTATT (SEQ. ID. NQ. TTGCTGTTGCTCC (Bam HI) 54) D 131B 5′NC AGGCAGACCACATATGTG (SEQ. ID. NO. 55) R 119 B 1GGTGCACTCCTGACCAAGCC (SEQ. ID. NO. 56) D 118 B 1 ATTGGCTGCCACTTTGTTC(SEQ. ID. NO. 57) R 117 B 1 ACCCTCATACGTCACCACAA (SEQ. ID. NO. C 58) R116 B 1 GCGGTGGACCACATTAGGAT (SEQ. ID. NO. TATC 59) D 115 B 1CATGATATGTCACCATCTG (SEQ. ID. NO. 60) D 114 B 1 GTCATCCATAACGAGCTGG(SEQ. ID. NO. 61) R 112 B 2 AGCGGAATTCGAGGGGCGGC (SEQ. ID. NO.ATAAAGAACCAGG (EcoRI) 62) R 111 B 2 GCGCTGAATTCGGATCACAA (SEQ. ID. NO.GCTCAGAGGCTATGCC 63) (EcoRI) D 110 B 2 GTATAACGGATCCACATCTC (SEQ. ID.NO. CCCTTACCTC (Bam HI) 64) D 109 B 2 TAACCTGGATCCTTATGCCG (SEQ. ID. NO.CCCCTCTTAG (Bam HI) 65) D 108 B 1 AAATTGGATCCTGTGTCGGG (SEQ. ID. NO.TGGAATGAATAACATGTC 66) (BamHI) R 107 B 1 ATCGGCAGATCTGATAGAGC (SEQ. ID.NO. GGGGACTTGCCGGATCC 67) D 101 B 2 TACCCTGCCCGCGCCCATAC (SEQ. ID. NO.TTTTGATG 68) R 100 B 1 GGCTGAGATCTGGTTCGGGT (SEQ. ID. NO. CGCCAAGAAGGTG(Bgl II) 69) R 99 B 2 TACAGATCTATACAACTTAA (SEQ. ID. NO. CAGTCGG (BglII) 70) R 98 B 2 GCGGCAGATCTCACCGACAC (SEQ. ID. NO. CATTAGTAC (Bgl II)71) D 97 S 1 CCGTCGGATCCCAGGGGCTG (SEQ. ID. NO. CTGTCCTG (Bam HI) 72) R96 B 2 AAAGGAATTCAAGACCAGAG (SEQ. ID. NO. GTAGCCTCCTC (EcoRI) 73) D 95 B2 GTTGATATGAATTCAATAAC (SEQ. ID. NO. CTCGACGG 74) R 94 B 3′NCTTTGGATCCTCAGGGAGCGC (SEQ. ID. NO. GGAACGCAGAAATGAG 75) (BamHI) D 90 B 2TCACTCGTGAATTCCTATAC (SEQ. ID. NO. TAATAC (EcoRI) 76) R 89 B 3′NCTTTGGATCCTCAGGGAGCGC (SEQ. ID. NO. GGAACGCAGAAATG (BamHI) 77) R 88 B 1TGATAGAGCGGGACTTGCCG (SEQ. ID. NO. GATCC (BamHI) 78) R 87 B 1TTGCATTAGGTTAATGAGGA (SEQ. ID. NO. TCTC 79) D 86 B 1ACCTGCTTCCTTCAGCCTGC (SEQ. ID. NO. AGAAG 80) R 81 B 1GCGGTGGATCCGCTCCCAGG (SEQ. ID. NO. CGTCAAAAC (BamHI) 81) D 80 B 1GGGCGGATCGAATTCGAGAC (SEQ. ID. NO. CCTTCTTGG (EcoRI) 82) R 79 B 1AGGATGGATCCATAAGTTAC (SEQ. ID. NO. CGATCAG (BamHI) 83) D 78 B 1GGCTGGAATTCCTCTGAGGA (SEQ. ID. NO. CGCCCTCAC (EcoRI) 84) R 77 B 1GCCGAAGATCTATCGGACAT (SEQ. ID. NO. AGACCTC (Bgl II) 85) R 76 B 2CAGACGACGGATCCCCTTGG (SEQ. ID. NO. ATATAGCCTG (BamHI) 86) D 75 B 5′NCGGCCGAATTCAGGCAGACCA (SEQ. ID. NO. CATATGTGGTCGATGCCATG 87) (EcoRI) D 72B 1 GCAGGTGTGCCTGGATCCGG (SEQ. ID. NO. CAAGT (BamHI) 88) R 71 B 1GTTAGAATTCCGGCCCAGCT (SEQ. ID. NO. GTGGTAGGTC (EcoRI) 89) D 63 B 1CCGTCCGATTGGTCTGTATG (SEQ. ID. NO. CAGG 90) D 61 B 1TACCAGTTTACTGCAGGTGT (SEQ. ID. NO. GC 91) D 60 B 1 CAAGCCGATGTGGACGTTGT(SEQ. ID. NO. CG 92) R 59 B 2,3 GGCGCTGGGCCTGGTCACGC (SEQ. ID. NO. CAAG93) D 50 B 1 GCAGAAACTAGTGTTGACCC (SEQ. ID. NO. AG 94) R 49 B 2TAGGTCTACGACGTGAGGCA (SEQ. ID. NO. AC 95) R 48 B 1 TACAATCTTTCAGGAAGAAG(SEQ. ID. NO. G 96) R 47 B 1 CCCACACTCCTCCATAATAG (SEQ. ID. NO. C 97) D46 B 1 GATAGTGCTTTGCAGTGAGT (SEQ. ID. NO. ACCG 98)

The abbreviations to the left of the sequences represent the following:R and D refer to reverse and forward primers, respectively; B and Srefer to sequences derived from the Burma-121 Strain of Hepatitis E andthe SAR-55 Strain of Hepatitis E, respectively; 5′NC and 3′NC refer to 5prime and 3 prime non-coding regions of the HEV genome, respectively;and 1, 2 and 3 refer to sequence derived from open reading frames 1, 2or 3, respectively. The symbol ( ) to the right of some sequences shownindicates insertion of an artificial restriction site into thesesequences.

For cloning of PCR fragments, EcoRI, BamHI, or BglII restriction sitespreceded by 3-7 nt were added to the 5′ end of primers.

RT-PCR. The usual 100-μl RT-PCR mixture contained template, 10 mMTris-HCL (ph 8.4), 50 mM KCl, 2.5 mM MgCl₂, all four dNTPs (each at 0.2mM), 50 pmol of direct primer, 50 pmol of reverse primer, 40 units ofRNasin (Promega), 16 units of avian myeloblastosis virus reversetranscriptase (Promega), 4 units of AmpliTaq (Cetus), under 100 μl oflight mineral oil. The mixture was incubated 1 h at 42° C. and thenamplified by 35 PCR cycles; 1 min at 94° C., 1 min at 45° C., and 1 minat 72° C. The PCR products were analyzed on 1% agarose gels.

Cloning of PCR Fragments. PCR fragments containing restriction sites atthe ends were digested with EcoRI and BamHI or EcoRI and BglIIrestriction enzymes and cloned in EcoRI/BamHI-digested pBR322 or pGEM-3Z(Promega). Alternatively, PCR fragments were cloned into pCR1000(Invitrogen) using the TA cloning kit (Invitrogen).

Sequencing of PCR Fragments and Plasmids. PCR fragments were excisedfrom 1% agarose gels and purified by Geneclean (Bio 101, La Jolla,Calif.). Double-stranded PCR fragments were sequenced by using Sequenase(United States Biochemical) as described in Winship, P. R. (1984),Nucleic Acids Rev., 17:1266. Double-stranded plasmids purified throughCsCl gradients were sequenced with a Sequenase kit (United StatesBiochemical).

Computer Analysis of Sequences. Nucleotide sequences of HEV strains werecompared using the Genetics Computer Group (Madison, Wis.) softwarepackage (Devereaux, J. et al. (1984), Nucleic Acids Rev., 12:387-395,version 7.5, on a VAX 8650 computer (at the National Cancer Institute,Frederick, Md.)).

EXAMPLE 2 Construction of a Recombinant Expression Vector, P63-2

A plasmid containing the complete ORF-2 of the genome of HEV strainSAR-55, Tsarev, S. A. et al. (1992), Proc. Natl. Acad. Sci. USA,89:559-563), was used to obtain a restriction fragment NruI-BglII. NruIcut the HEV cDNA five nucleotides upstream of the ATG initiation codonof ORF-2. An artificial Bgl II site previously had been placed at the 3′end of HEV genome just before the poly A sequence (Tsarev, S. A. et al.(1992), Proc. Natl. Acad. Sci. USA, 89:559-563). To insert this fragmentinto pBlueBac-Transfer vector (Invitrogen) a synthetic polylinker wasintroduced into the unique NheI site in the vector. This polylinkercontained Bln I and Bgl II sites which are absent in both HEV cDNA andpBlueBac sequences. The NruI-BglII ORF-2 fragment was inserted in BlnI-BglII pBlueBac using an adapter as shown in FIG. 1.

EXAMPLE 3 Expression of P63-2 in SF9 Insect Cells

p63-2 and AcMNPV baculovirus DNA (Invitrogen) were cotransfected intoSF9 cells (Invitrogen) by the Ca precipitation method according to theInvitrogen protocol—By following this protocol; the AcMNPV baculovirusDNA can produce a live intact baculovirus which can package p63-2 toform a recombinant baculovirus. This recombinant baculovirus wasplaque-purified 4 times. The resulting recombinant baculovirus 63-2-IV-2was used to infect SF9 cells.

SDS-PAGE and Western blot. Insect cells were resuspended in loadingbuffer (50 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromphenol blueand 10% glycerol) and SDS-polyacrylamide gel electrophoresis wasperformed as described, Laemmli, U.K. (1970), Nature, 227:680. Gels werestained with coomassie blue or proteins were electroblotted onto BA-85nitrocellulose filters (Schleicher & Schuell). After transfer,nitrocellulose membranes were blocked in PBS containing 10% fetal calfserum and 0.5% gelatin. As a primary antibody, hyperimmune serum ofchimpanzee-1313 diluted 1:1000 was used. As a secondary antibody,phosphatase-labeled affinity-purified goat antibody to human IgG(Kirkegaard & Perry Laboratories, Inc.) diluted 1:2000 was used. Filterswere developed in Western blue stabilized substrate for alkalinephosphatase (Promega). All incubations were performed in blockingsolution, and washes were with PBS with 0.05% Tween-20 (Sigma).

Expression of HEV ORF-2. The major protein synthesized in SF9 cellsinfected with recombinant baculovirus 63-2-IV-2 was a protein with anapparent molecular weight of 74 KD (FIG. 2A, lane 3). This size is alittle larger than that predicted for the entire ORF-2 (71 KD). The sizedifference could be due to glycosylation of the protein since there isat least one potential site of glycosylation (Asn-Leu-Ser) in theN-terminal part. This protein was not detected in noninfected cells(FIG. 2A, lane 1) or in cells infected with wild-type nonrecombinantbaculovirus (FIG. 2A, lane 2). In the latter case, the major proteindetected was a polyhedron protein. When the same lysates were analyzedby Western blot (FIG. 2B) with serum of chimp-1313 (hyperimmunized withHEV), only proteins in the recombinant cell lysate reacted (lane 3) andthe major band was again represented by a 74 KD protein (FIG. 2B). Minorbands of about, 25, 29, 35, 40-45 and 55-70 kDa present in theCoomassie-stained gel (FIG. 2A, lane 3) also reacted with serum in theWestern blot (FIG. 2B, lane 3). Some of the bands having molecularweights higher than 74 KD result from different extents of glycosylationwhile the lower molecular weight bands could reflect processing and/ordegradation. Serum drawn from Chimp-1313 prior to inoculation with HEVdid not react with any of the proteins by Western blot.

EXAMPLE 4 Immunoelectron Microscopy of Recombinant Infected SF9 Cells

5×10⁶ recombinant infected SF9 cells were sonicated in CsCl (1.30 g/ml)containing 10 mM Tris-HCl, pH 7.4, 0.3% sarcosyl and centrifuged 68 h,at 40,000 rpm (SW60Ti). 50 ul of the fraction, which had the highestELISA response and a buoyant density of 1.30 g/ml was diluted in 1 mlPBS and 5 ul of chimp-1313 hyperimmune serum was added. The hyperimmuneserum was prepared by rechallenging a previously infected chimp with asecond strain of hepatitis E (Mexican HEV). Samples were incubated 1 hat room temperature and then overnight at 4° C. Immune complexes wereprecipitated using a SW60Ti rotor at 30,000 rpm, 4° C., 2 h. Pelletswere resuspended in distilled water, negatively stained with 3% PTA,placed on carbon grids and examined at a magnification of 40,000 in anelectron microscope EM-10, Carl Zeiss, Oberkochen, Germany.

Detection of VLPs. Cell lysates from insect cells infected withwild-type or recombinant baculovirus 63-2-IV-2 were fractionated by CsCldensity centrifugation. When fractions of the CsCl gradient from therecombinant infected insect cells were incubated with Chimp-1313hyperimmune serum, two kinds of virus-like particles (VLP) covered withantibody were observed in the fraction with buoyant density of 1.30g/ml: first (FIGS. 3A, 3A′, 3A″ amd 3A′″), antibody covered individualparticles that had a size (30 nm) and morphological structure suggestiveof HEV, second (FIG. 3B), antibody-coated aggregates of particlessmaller than HEV (about 20 nm) but which otherwise resembled HEV. DirectEM showed the presence of a very heterogenous population of objectsincluding some of 30 and 20 nm in diameter respectively, which lookedlike virus particles but, in the absence of bound antibody, could not beconfirmed as HEV. A number of IEM experiments suggested that at leastsome of the protein(s) synthesized from the ORF-2 region of the HEVgenome, had assembled into a particulate structure. It was observed thatinsect cells at a later stage of infection, when the proportion ofsmaller proteins was higher, consistently gave better results in ELISA.Therefore, unfractionated lysates of recombinant insect cells from alater stage of infection were used as antigen in ELISA in subsequenttests.

EXAMPLE 5 Detection by ELISA Based on Antigen from Insect CellsExpressing Complete ORF-2 of Anti-HEV Following Infection with DifferentStrains of HEV

5×10⁶ SF9 cells infected with 63-2-IV-2 virus were resuspended in 1 mlof 10 mM Tris-HCl, pH 7.5, 0.15M NaCl then were frozen and thawed 3times. 10 ul of this suspension was dissolved in 10 ml of carbonatebuffer (pH 9.6) and used to cover one flexible microtiter assay plate(Falcon). Serum samples were diluted 1:20, 1:400 and 1:8000, or 1:100,1:1000 and 1:10000. The same blocking and washing solutions as describedfor the Western blot were used in ELISA. As a secondary antibody,peroxidase-conjugated goat IgG fraction to human IgG or horse radishperoxidase-labelled goat anti-Old or anti-New World monkeyimmunoglobulin was used. The results were determined by measuring theoptical density (O.D.) at 405 nm.

To determine if insect cell-derived antigen representing a Pakistanistrain of HEV could detect anti-HEV antibody in cynomolgus monkeysinfected with the Mexican strain of HEV, 3 monkeys were examined (FIG.4). Two monkeys cyno-80A82 and cyno-9A97, were infected with fecescontaining the Mexico '86 HEV strain (Ticehurst, J. et al. (1992), J.Infect. Dis., 165:835-845) and the third monkey cyno-83 was infectedwith a second passage of the same strain. As a control, serum samplesfrom cyno-374, infected with the Pakistani HEV strain SAR-55, weretested in the same experiment. All 3 monkeys infected with the Mexicanstrain seroconverted to anti-HEV. Animals from the first passageseroconverted by week 15 and from the second passage by week 5.Interestingly, the highest anti-HEV titer among the 4 animals, was foundin cyno-83, inoculated with the second passage of the Mexican strain.Cynos inoculated with the first passage of the Mexican strain developedthe lowest titers while those inoculated with the first passage of thePakistani strain developed intermediate titers.

EXAMPLE 6 Specificity of Anti-HEV ELISA Based on Antigen from InsectCells Expressing Complete ORF-2

To estimate if the ELISA described here specifically detected anti-HEVto the exclusion of any other type of hepatitis related antibody, serumsamples of chimps were analyzed, in sets of four, infected with theother known hepatitis viruses (Garci, P. et al. (1992), J. Infect. Dis.,165:1006-1011; Farci, P. et al. (1992), Science (in press); Ponzetto, A.et al. (1987) J Infect. Dis., 155: 72-77; Rizzetto; m.et al. (1981)Hepatology 1: 567-574 ; reference for chimps—1413, 1373, 1442, 1551(HAV); and for chimps—982, 1442, 1420, 1410 (HBV); is unpublished datafrom Purcell et al) (Table 1). Samples of pre-inoculation and 5 week and15 week post-inoculation sera were analyzed in HEV ELISA at serumdilutions of 1:100, 1:1000 and 1:10000. None of the sera from animalsinfected with HAV, HBV, HCV and HDV reacted in the ELISA for HEVantibody, but all 4 chimps inoculated with HEV developed the IgM and IgGclasses of anti-HEV.

TABLE 1 Serological assay of anti-HEV antibody in chimpanzees infectedwith different hepatitis viruses (Hepatitis A, B, C, D, E) week ofseroconversion weeks post-inoculation inoculated for inoculated preserum5 15 20/25 chimp virus virus IgG IgM IgG IgM IgG IgM IgG IgM Chimp-1413HAV 5 — — — — — — Chimp-1373 HAV 7 — — — — — — Chimp-1442 HAV 5 — — — —— — Chimp-1451 HAV 5 — — — — — — Chimp-982 HBV 3 — — — — — — Chimp-1442HBV 7 — — — — — — — — Chimp-1420 HBV 9 — — — — — — Chimp-1410 HBV 5 — —— — — — Chimp-51 HCV 10 — — — — — — Chimp-502 HCV 12 — — — — — —Chimp-105 HCV 28 — — — — — — Chimp-793 HCV 13 — — — — — — Chimp-904 HDV8 — — — — — — Chimp-814 HDV 7 — — — — — — Chimp-800 HDV 10 — — — — — —Chimp-29 HDV 10 — — — — — — — — Chimp-1310 HEV 5 — — 1:10,000 1:1001:10,000 — Chimp-1374 HEV 3 — — 1:8000 —* 1:8000 — Chimp-1375 HEV 3 — —1:8000 1:400 1:400 — Chimp-1313 HEV1st°** 5 — — 1:10,000 1:100 1:1000 —Chimp-1313 HEV2nd°** 0.5 1:100 — 1:10,000 — 1:10,000 — *Chimp-1374 waspositive for IgM anti-HEV three and four weeks post-inoculation (seeFIG. 5) **Chimp-1313 was inoculated with HEV twice. 1st inoculation withpooled samples of 7 Pakistani patients. 2nd inoculation 45 months laterwith Mexican strain of HEV.

EXAMPLE 7 Determination of the Host Range of the SAR-55 Strain of HEV inNon-Human Primates

Different primate species were inoculated intravenously with a standardstool suspension of HEV and serial serum samples were collected tomonitor for infection. Serum ALT levels were determined as an indicatorof hepatitis while seroconversion was defined as a rise in anti-HEV. Theresults were compared with those obtained in cynomolgus monkeys andchimpanzees.

Both rhesus monkeys inoculated with HEV (Table 2) demonstrated veryprominent peaks of alanine aminotransferase activity as well as a stronganti-HEV response. The peak of alanine aminotransferase activity wasobserved on day 35 for both animals, and seroconversion occurred on day21. The maximum titer of anti-HEV was reached on day 29. Both Africangreen monkeys used in this study (Table 2) developed increased alanineaminotransferase activity and anti-HEV. Although African green money 230died 7 weeks after inoculation, proof of infection was obtained beforethat time. Peak alanine aminotransferase activity for monkey 74 exceededthe mean value of preinoculation sera by about three times and formonkey 230 by about five times. Peaks of alanine aminotransferaseactivity and seroconversion appeared simultaneously on days 28 and 21 inmonkeys 74 and 230, respectively.

TABLE 2 Biochemical and serologic profiles of HEV infection in eightprimate species. Alanine aminotransferase (units/L) Anti-HEV IgG Pre-Day first inoculation, detected Maximum Animal mean (SD) Day Value(titer) titer Chimpanzee 1374 51 (12) 27 114 27(1:400) 1:8000 1375 41(14) 27 89 27(1:400) 1:8000 Cynomolgus monkey 374* 46 (20) 26 60819(1:400) 1:8000 381* 94 (19) 35 180 28(1:20) 1:8000 Rhesus monkey 72643 (6) 35 428 21(1:20) 1:8000 938 29 (10) 35 189 21(1:20) 1:8000 Africangreen monkey 74 72 (21) 28 141 28(1:400) 1:8000 230 102 (45) 21 33421(1:8000) 1:8000 Pigtail macaque 98 37 (8) 21 47 21(1:400) 1:8000 99 41(6) 28 59 21(1:400) 1:8000 Tamarin 616 28 (7) 70 41 — 636 19 (4) 7, 5630 — Squirrel monkey 868 90 (35) 40 355 41(1:20) 1:20 869 127 (63) 42679 35(1:20) 1:20 Owl monkey 924 41 (7) 35 97 21(1:20) 1:8000 925 59 (6)49, 91† 78,199† 21(1:20) 1:8000 NOTE. -, no anti-HEV detected.*Previously studied using fragments of HEV proteins expressed inbacteria as antigen [18]. †Biomodal elevation of alanineaminotransferase. SD = standard deviation.

Pigtail macaque 99 demonstrated an increase in alanine aminotransferaseactivity >3 SD above the mean value of preinoculation sera, whilepigtail macaque 98 did not. However, both monkeys seroconverted on day21 and the anti-HEV titers were equivalent to those of the chimpanzeesand Old World monkeys. Because of the low peak alanine aminotransferasevalues in the pigtail macaques, the possibility of immunization insteadof infection with HEV cannot be completely ruled out. However,immunization is unlikely for several reasons. First, immunization ineither of 2 tamarins, which are only one-fourth as large as the pigtailmacaques but received the same amount of inoculum was not observed.Second, it is well known that the amount of HEV excreted in feces isusually very small, and 0.5 mL of the 10% suspension of feces used inthis study is unlikely to contain an amount of antigen sufficient toimmunize an animal, especially when inoculated intraveneously.

Neither tamarin inoculated in this study had a significant rise inalanine aminotransferase activity or development of anti-HEV (Table 2).Therefore, these animals did not appear to be infected. The squirrelmonkeys did develop anti-HEV, but at significantly lower levels thanchimpanzees or Old World monkeys (Table 2). In addition, seroconversionoccured later in other animals. Squirrel monkey 868 seroconverted on day41 and 869 on day 35. The anti-HEV titer was not >1:20 at any timeduring >3 months of monitoring and clearly was waning in both animalsafter reaching a peak value on days 47-54. However, the increases inalanine aminotransferase activity were rather prominent in both animalsand were temporally related to the time of seroconversion.

The owl monkeys responded to HEV infection about as well as the OldWorld monkey species (Table 2). Both owl monkeys seroconverted on day 21and by day 28 the anti-HEV titer had reached a value of 1:8000. Alanineamino-transferase activity peaked on day 35 in owl monkey 924 but notuntil day 49 in monkey 925.

EXAMPLE 8 Detection of IgM and IgG Anti-HEV in Chimps

In both chimps, the serum ALT levels increased about 4 weekspost-inoculation (Table 2, FIG. 5). Both chimps seroconverted at thetime of ALT enzyme elevation or earlier (FIG. 5A, 5C). Levels of IgManti-HEV also were determined for the chimps. In chimp-1374, the titerof IgM anti-HEV (FIG. 5B) was not as high as the IgG titer (FIG. 5A) andwaned over two weeks. Although both IgG and IgM antibodies were firstdetected for this animal on day 20, the titer of IgM anti-HEV was thehighest while the titer of IgG was the lowest on that day, but then roseand stayed approximately at the same level for more than three months.In chimp-1375, only IgM anti-HEV was detected on day 20 (FIG. 5D). Thetiter was higher than in chimp-1374 and IgM anti-HEV was detected duringthe entire period of monitoring. IgG anti-HEV was first observed in thisanimal on day 27 (FIG. 5C) and remained at approximately the same levelthroughout the experiment.

EXAMPLE 9 Comparison of ELISA Based on Complete ORF-2 Protein Expressedin Insect Cells with that Based on Fragments of Structural ProteinsExpressed in E. coli.

To estimate if expression of the complete ORF-2 region of the HEV genomein eukaryotic cells had any advantage over expression of fragments ofstructural proteins in E. coli, we used the former antigen in ELISA toretest cynomolgus monkey sera that had been analyzed earlier (Tsarev, S.A. et al. (1992), Proc. Natl. Acad. Sci USA, 89:559-563; and Tsarev, S.A. et al. (1993) J. Infect. Dis. (167:1302-1306)), using the antigenfragments expressed in bacteria (Table 3).

TABLE 3 Comparison of ELISA based on antigen from insect cellsexpressing complete ORF-2 with that based on antigen from E.coliexpressing fragments of structural proteins antigen derived from antigenderived from insect cells bacterial cells (Complete ORF-2) (Portion ofORF-2)* anti-HEV day anti- first HEV first detected max. Cyno # detectedday titer titer Cyno-376 28 21 1:400 1:8000 Cyno-369 54 40 1:100 1:8000Cyno-374 19 19 1:400 1:8000 Cyno-375 26 26 1:400 1:8000 Cyno-379 21 191:100 1:8000 Cyno-381 28 28 1:400 1:8000 *The sera were also tested withless sensitive ORF-3 antigen [..]. Tsarev, S.A. et al. (1993), J.Infect. Dis. (167:1302-1306)

For 3 of the 6 monkeys examined by ELISA, the antigen expressed ininsect cells detected seroconversion earlier than the antigen expressedin E. coli. Using the insect cell-derived antigen, we were able todetect anti-HEV antibody in sera from all six monkeys at the highestdilution tested (1:8000). With E. coli-cell derived antigen (BurmaStrain) no information about anti-HEV titers were obtained, since allsera were tested only at a dilution of 1:100 (Tsarev, S A et al (1992)Proc. Nat. Acad. Sci. USA; 89:559-563; Tsarev et al. (1993) J. Infect.Dis. (167:1302-1306)).

In another study, hepatitis E virus, strain SAR-55 was serially dilutedin 10-fold increments and the 10⁻¹ through 10⁻⁵ dilutions wereinoculated into pairs of cyno-molgus monkeys to titer the virus. Theserum ALT levels were measured to determine hepatitis and serum antibodyto HEV was determined by the ELISA method of the present invention (datain figures) or by Genelab's ELISA (three ELISAs, each based on one ofthe antigens designated 4-2, 3-2 and 612 in Yarbrough et al. (J. Virol.,(1991) 65:5790-5797) (data shown as positive (+) or negative (−) test atbottom of FIGS. 6a-g). All samples were tested under code.

The ELISA method of the present invention detected seroconversion to IgGanti-HEV in all cynos inoculated and all dilutions of virus.

In contrast, Genelab's results were strikingly variable, as summarizedbelow.

TABLE 4 ELISA of Dilution Present of Virus Genelab's ELISA Invention10⁻¹ did not test positive 10⁻² positive for both animals, positivelimited duration 10⁻³ negative for both animals positive 10⁻⁴ Cyno 389:positive for IgM positive and IgG Cyno 383: negative positive 10⁻⁵ Cyno386: negative positive Cyno 385: positive positive

Since Cyno 385 (10⁻⁵) was positive in ELISA tests both by Genelabs andthe present invention, the 10⁻⁴ (ten times more virus inoculated) and10⁻³ (100 times more virus inoculated) would also have been expected tobe positive. The present invention scored them as positive in contrastto Genelab's ELISA test which missed both positives at 10⁻³ and one at10⁻⁴ even though the ALT levels of Cyno 383 and 393 suggested activehepatitis. Therefore, the data support the advantages of the presentELISA method over the prior art methods of detecting antibodies to HEV.

EXAMPLE 10 Comparison of ELISAs Based on Recombinant ORF-2 AntigensConsisting of Either a 55 kDa Protein Expressed from the Complete ORF-2Region of the Pakistani SAR-55 Strain of HEV or of Shorter Regions ofORF-2 Expressed as Fusion Proteins in Bacteria

As described in Example 3 and as shown in FIGS. 2A and 2B, a number ofproteins of varying molecular weights are expressed in insect cellsinfected with the recombinant baculovirus containing the complete ORF-2.A protein with a molecular weight of approximately 55 kDa was partiallypurified from 5×10⁸ SF-9 cells harvested seven days post-inoculation asfollows: The infected cells were centrifuged, resuspended in 10 ml of 10mM Tris-HCl (pH 8.0), 50 mM NaCl, containing 40 μg/ml ofphenylmethylsulfonyl fluoride (Sigma, St. Louis, Mo.), sonicated todisrupt the cells and the lysate was centrifuged at 90,000×g at 4° C.for 30 min. The supernatant was loaded onto a DEAE-sepharose CL-6B(Pharmacia, Uppsala, Sweden) column equilibrated with 10 mM Tris-HCl (pH8.0), 50 mM NaCl. The column was washed with loading buffer and the 55kDa protein was eluted in 10 mM Tris-HCl (pH 8.0) 250 mM NaCl. Fractionscontaining the 55 kDa protein were combined and the protein wasprecipitated by addition of 3 g of (NH₄)₂SO₄ to 10 ml of the proteinsolution. The protein pellet was dissolved in 10 mM Tris-HCl (pH 8.0),50 mM NaCl. The 55 kDa protein was then used as the insectcell-expressed HEV antigen in ELISA in comparison testing against ELISAsbased on either one of two HEV antigens expressed in bacteria, (3-2(Mexico) (Goldsmith et al., (1992) Lancet, 339:328-331) or SG3 (Burma)(Yarbough et al., (1993) Assay development of diagnostics tests forhepatitis E. In “International Symposium on Viral Hepatitis and LiverDisease. Scientific program and abstract volume.” Tokyo:VHFL, p 87,Abstract #687). These bacterial antigens were fusion proteins of the 26kDa glutathione-S-transferase (GST) and either the antigenic sequence3-2 (M) consisting of 42 amino acids located 6 amino acids upstream ofthe C-terminus of ORF-2 (Yarbough et al., (1991) J. Virol.,65:5790-5797) or the 327 C-terminal amino acids of ORF-2 (Yarbough etal., (1993)). The ELISAs were carried out as follows.

Sixty ng per well of the 55 kDa protein or 200 ng per well of the fusionantigens in carbonate buffer (pH 9.6) were incubated in wells of apolystyrene microtiter assay plate (Dynateck, S. Windham, Me.) for 2 hat 37° C. Plates were blocked with PBS containing 10% fetal calf serumand 0.5% gelatin. Serum samples from cynomolgus monkeys inoculatedintravenously (note: cynos 387 and 392 were inoculated orally) with adilution of feces containing the SAR-55 strain of HEV ranging from 10⁻¹through 10⁻⁸ as indicated in Table 5 and FIGS. 7A-7J and 8A-8D werediluted 1:100 in blocking solution. Peroxidase-conjugated goatanti-human IgM (Zymed, San Francisco, Calif.) diluted 1:1000 or 1:2000,or peroxidase-labelled goat anti-human immunoglobulin diluted 1:1000 wasused as the detector antibody.

In all of the ELISA tests except those for the two orally inoculatedmonkeys, cyno-387 and cyno-392, the 55 kDa and the fusion antigens weretested concurrently in the same laboratory so that the only variable wasthe antigen used. Criteria for scoring positive reactions in anti-HEVELISA with the 55 kDa antigen were an optical density value≧0.2 andgreater than twice that of a pre-inoculation serum sample for the sameanimal. In addition, since both antigens expressed in bacteria werefusion proteins with GST, the optical density of a sample tested withthese antigens had to be 3 times higher than that obtained withnon-fused GST in order to be considered positive (Goldsmith et al.,(1992)).

RESULTS

Both cynomolgus monkeys (377, 378) inoculated with the 10⁻¹ dilution ofthe standard HEV fecal suspension had a pronounced increase in ALTactivity at 4-5 weeks post-inoculation, indicative of hepatitis (Table5, FIGS. 7A and 7B).

TABLE 5 Summary of biochemical and serological events occurring incynomolgus monkeys after inoculation with 10⁻¹ to 10⁻⁸ dilutions of thestandard stock of the SAR-55 HEV inoculum. weeks post- inoculationanti-HEV peak weeks post- was detected inoculation anti-HEV was CynoDilution ALT with 55 kDa detected with fusion antigen of viral stockpre-inoculation peak antigen (U/L) IgG IgM inoculum week mean (SD)^(¶)value IgG IgM SG3 3-2(M) SG3 3-2(M) 377 10⁻¹ 76 (39) 5 264  4-15^(†)3-7  4-10 4-5 3-4 3-5 378 10⁻¹ 50 (9) 4 285  4-15  — — — — — 394 10⁻² 62(14) 4 89  3-15 3-10 — 4-6 — — 395 10⁻² 121 (21) 15 314  5-15 — — — — —380 10⁻³ 89 (20) 1 135  5-15* — 6-15 — — — 383 10⁻³ 29 (8) 4 77  5-155-13 — — — — 389 10⁻⁴ 60 (7) 15 114  6-15 6-8  — — — — 393 10⁻⁴ 41 (4) 587  6-15 — — — — — 385 10⁻⁵ 59 (32) 7 56 11-15 — —  7-15 — — 386 10⁻⁵ 31(4) 4 34  8-15 8-13 — — — — 397 10⁻⁶ 60 (4) 8 94 — — — — — — 398 10⁻⁶ 36(3) 2 55 — — — — — — 399 10⁻⁷ 102 (16) 2 93 — — — — — — 400 10⁻⁷ 57 (4)9 188 — — — — — — 403 10⁻⁸ 33 (3) 2-3 49 — — — — — — 406 10⁻⁸ 56 (4) 273 — — — — — — 387 10⁻¹ (oral)^(§) 32 (4) 4 38 — — ND — ND — 392 10⁻¹(oral)^(§) 49 (6) 3 70 — — ND — ND — ^(¶)ALT mean and standard deviation(SD) values of pre-inoculation sera. ^(†)The experiment was terminatedafter 15 weeks. *The OD values of pre-inoculation sera of Cyno-380, whentested by ELISA with 55 kDa antigen, were twice as high as the meanvalue of pre-inoculation sera for other cynomolgus monkeys. ^(§)AllELISA tests except for Cyno-387 and Cyno-392 were performed in the sameexperiments. — not detected. ND - not done.

All 3 antigens tested detected IgM anti-HEV in samples taken fromcyno-377 3 weeks post-inoculation (Table 5, FIG. 8A), but IgM anti-HEVwas not detected in any samples from the second animal, cyno-378. IgGanti-HEV was detected in both animals with the 55 kDa-based ELISA, butonly in cyno-377 with the ELISA based on fusion antigens. Values of ODfor IgG anti-HEV were significantly higher than those for IgM anti-HEV.ELISA values obtained with the 55 kDa antigen were also significantlyhigher than those obtained with either of the two fusion antigens (FIGS.7A and 7B). The patterns of the OD values observed in ELISA withantigens from the two sources also differed significantly. In the caseof ELISA based on the fusion antigens, positive signals reached amaximum shortly after seroconversion and then waned during the 15 weeksof the experiment. In ELISA based on the 55 kDa antigen, the positivesignal reached a maximum shortly after seroconversion and remained atapproximately the same high level throughout the experiment (15 weeks).

Elevation in ALT activities in both monkeys (394 and 395) inoculatedwith a 10⁻² dilution of the standard HEV stool suspension wassignificantly less pronounced at the expected time of hepatitis than inanimals inoculated with the ten-fold higher dose (Table 5, FIGS. 7C and7D). Cyno-395 actually had higher ALT activities prior to inoculation aswell as at 15 weeks post-inoculation. The latter was probably notrelated to HEV infection. Weakly positive IgM anti-HEV was detected onlyin cyno-394 (FIG. 8B) and only with ELISA based on the 55 kDa antigen.Both animals were infected, however, since IgG anti-HEV seroconversionwas detected in both animals. In cyno-394, anti-HEV IgG was firstdetected by the 55 kDa antigen at week 3 and one week later with the3-2(M) antigen. The SG3 (B) antigen did not detect seroconversion incyno-395 and anti-HEV IgG was detected only with the 55 kDa antigen.Anti-HEV tended to diminish in titer with time in this animal.

Cyno-380 and cyno-383 were inoculated with a 10⁻³ dilution of thestandard HEV fecal suspension (Table 5, FIGS. 7E7F, 8C). Cyno-380 hadfluctuating ALT activities before and after inoculation; therefore, ALTlevels could not be used to document hepatitis E in this animal. InCyno-383, a slight rise of ALT activities was observed (FIG. 7F), whichwas coincident with seroconversion and, therefore, might be due to mildhepatitis E. IgM Anti-HEV was not detected in sera from cyno-380 withany of the three antigens. Cyno-380 seroconverted for IgG anti-HEV whentested by ELISA with SG3 (B) but not with 3-2(M) antigen. This animalhad preexisting IgG anti-HEV when tested with the 55 kDa antigen, butthere was a large increase in IgG anti-HEV starting at week 5 (FIG. 7E).Identification of preexisting antibody was reported earlier in sera fromanother cynomolgus monkey [Ticehurst et al., (1992) J. Infect Dis.,165:835-845; Tsarev et al., (1993) J. Infect. Dis., 168:369-378].Seroconversion occured at the expected time but the levels of IgGanti-HEV in samples from cyno-383 remained low and detectable only withthe 55 kDa antigen.

Cyno-389 and cyno-393 were inoculated with a 10⁻⁴ dilution of thestandard HEV fecal suspension (FIGS. 7G, 7H, 8D, Table 5). Neitheranimal had a significant rise in ALT activities, although the timing ofa small but distinct ALT peak in sera of cyno-393 at week 5 (FIG. 7H)suggested borderline hepatitis. ELISA based on the SG3 (B) or 3-2(M)antigens scored both animals as negative for HEV infection. In contrast,the 55 kDa antigen detected IgM anti-HEV in sera of cyno-389 at weeks6-8 post-inoculation (FIG. 8D) and IgG anti-HEV from week 6 through week15 in both animals.

Neither animal inoculated with the 10⁻⁵ dilution of the standard fecalsuspension developed a noticeable rise in ALT activities (FIG. 7I, 7J,Table 5), but, in cyno-386, IgM and IgG anti-HEV were detected at weeks8-13 and 8-15 respectively with the 55 kDa antigen (FIG. 7J, 8E).Cyno-385 anti-HEV IgG was detected with the 55 kDa and the 3-2(M)antigen but not with SG3 (B) antigen. In contrast to previous patterns,IgG anti-HEV was detected with a fusion antigen four weeks earlier andat higher levels than with the 55 kDa antigen.

None of the animals inoculated with dilutions of the standard HEV fecalsuspension in the range of 10⁻⁶-10⁻⁸ developed antibody to any of thethree HEV antigens. Increased ALT activities were not observed in thoseanimals, except for one rather prominent peak of ALT activity at week 9in cyno-400 (Table 5). However, the absence of seroconversion in thisanimal indicated that this peak probably was not related to HEVinfection.

With respect to the two cynomolgus monkeys (387 and 392) inoculatedorally with the 10⁻¹ dilution of the 10% fecal suspension, neithermonkey was infected since ALT levels did not rise and ELISA performedwith the 3-2(M) and 55 kDa antigens did not detect seroconversion to HEV(Table 5).

Finally, serological evidence for HEV infection was found in all animalsinoculated with decimal dilutions of the fecal suspension through 10⁻⁵;none of the animals receiving higher dilutions had such evidence.Prominent hepatitis, as defined by elevated ALT, was observed only inthe two monkeys infected with the 10⁻¹ dilution. Significantly lowerelevations of ALT activities were observed in some animals inoculatedwith higher dilutions of the fecal suspension while, in others,elevations were not found. Considered alone, these low ALT rises werenot diagnostic of hepatitis. However, the coincidence of seroconversionand appearance of these ALT peaks suggests the presence of mildhepatitis in these animals. Anti-HEV IgG seroconversion was detected inall animals inoculated with dilutions of fecal suspension ranging from10⁻¹-10⁻⁵. A tendency toward lower levels of IgG anti-HEV and delayedseroconversion was observed in animals inoculated with higher dilutionsof the stock.

In sum, the 55 kDa Pakistani ORF-2 antigen was more efficient thaneither the 3-2(M) or SG3 (B) antigen for detecting IgM and IgG anti-HEVin cynomolgus monkeys infected with the Pakistani strain of HEV. Forexample, for all animal sera except those from cyno-385, detection ofIgG or IgM anti-HEV by ELISA was more efficient with the 55 kDa antigenthan with either the 3-2(M) or SG3 antigen. ELISA with the 55 kDaantigen produced internally consistent and reproducible results,detecting IgG anti-HEV in all ten animals inoculated with a fecaldilution of 10⁻⁵ or lower. The magnitude of ELISA signals also decreasedas the inoculum was diluted. The fusion antigens did not produceconsistent results between the pairs of animals. Only one of each pairof animals inoculated with the 10⁻¹, 10⁻², 10⁻³, or 10⁻⁵ dilution showedseroconversion to IgG anti-HEV, and only a single seroconversion for IgManti-HEV was detected with these antigens. Neither of the animalsinoculated with the 10⁻⁴ dilution of the original inoculum seroconvertedto either of the two fusion antigens even though sera from one animal(cyno-393) had sustained high levels of anti-HEV IgG when assayed withthe 55 kDa antigen. Although, as discussed above, ELISA for IgM anti-HEVwas significantly less sensitive than ELISA for cynomolgus IgG anti-HEV,the 55 kDa antigen was able to detect anti-HEV IgM in more animals thanthe 3-2(M) or SG3 (B) antigen. In sum, a definitive conclusion about theinfectious titer of the Pakistani viral inoculum used in this studycould not be made with the combined data from the 3-2(M) and SG3 (B)based ELISA but could be made with data obtained with the 55 kDaPakistani ELISA alone.

With respect to cyno-385, the difference in anti-HEV IgG detectionbetween the two test results was four weeks. These data suggest thepresence of a distinct epitope in the 3-2(M) antigen recognized by thisanimal that is absent in the larger 55 kDa and SG3 (B) antigens. Whentotal insect cell lysate, which contained both complete ORF-2 (75 kDa)and 55 kDa proteins, was used as antigen to retest these samples, theresults were the same as when 55 kDa was used alone. This findingsuggests that the 55 kDa protein may not lack 3-2 epitope amino acidsbut rather that the conformation of the 3-2 epitope sequence differedamong all three antigens used in this study. Finally, it is interestingto note that despite the fact that antigen SG3 (B) contained a longerportion of ORF-2 and included the entire sequence of epitope 3-2, it didnot detect more positive sera than the 3-2(M) antigen.

EXAMPLE 11 Determination of the Infectious Titer of the HEV SAR-55 ViralStock BY RT-PCR

Knowledge of the infectious titer of inocula is critical forinterpretation of much of the data obtained in experimental infectionsof animal models. However, until now the infectious titer of an HEVviral stock has not been reported. Ten-fold dilutions of the fecalsuspension containing the SAR-55 strain of HEV were extracted and RT-PCRamplification was performed as follows to determine the highest dilutionin which HEV genomes could be detected. 200 ul of fecal suspension wasmixed with 0.4 ml of 1.5M NaCl plus 15% polyethylene glycol (PEG) 8000and kept overnite at 4° C. Pellets were collected by centrifugation for3 minutes in a microcentrifuge (Beckman, Palo Alto, Calif.) at 16,000 gand dissolved in 475 ul of solution containing 4.2M guanidinethiocyanate, 0.5% N-lauroylsarcosine, 0.25M TRIS-HCl (pH 8.0). 0.15 Mdithiothreitol (DTT), and 1.0 μg of tRNA. Fifty microliters of 1MTRIS-HCl (pH 8.0), 100 mM EDTA, and 10% SDS was then added. The RNA wasextracted twice with phenol-chloroform (1:1) at 65° C., followed bychloroform extraction at room temperature. To the upper phase, 250 μL of7.5 M ammonium acetate was added, and nucleic acids were precipitatedwith 0.6 mL of 2-propanol, washed with 75% ethanol, washed with 100%ethanol, and used for reverse transcription (RT) PCR.

For detection of the HEV genome, two sets of nested primers were usedthat represented sequences from the 3′ region (ORF-2) of the SAR-55genome. Primers for reverse transcription and the first PCR are shown asSEQ ID NO:99: GTATAACGGATCCACATCTCCCCTTACCTC and SEQ ID NO:100:TACAGATCTATACAACTTAACAGTCGG respectively. Primers for the second PCR areshown as SEQ ID NO: 101: GCGGCAGATCTCACCGACACCATTAGTAC and SEQ IDNO:102: TAACCTGGATCCTTATGCCGCCCCTCTTAG respectively. The RNA pellet wasdissolved in 20 μL of 0.05 M TRIS-HCl (pH 7.6), 0.06 M KCl, 0.01 MMgCl₂, 0.001 M DTT, 40 units of RNasin (Promega Biotec, Madison, Wis.),16 units of avian myeloblastosis virus reverse transcriptase (PromegaBiotec), and 10 pmol of reverse primer and incubated 1 hour at 42° C. To20 μL of reverse transcriptase mixture was added 100 μL of 0.01 MTRIS-HCl (pH 8.4), 0.05 M KCl, 0.0025 M MgCl₂, 0.0002 M each dNTP, 50pmol of direct primer, 50 pmol of reverse primer, and 4 units ofAmpliTaq (Perkin-Elmer Cetus, Norwalk, Conn.) under 100 μL of lightmineral oil. The HEV cDNA was amplified by 35 cycles of PCR:1 min at 94°C., 1 min at 55° C., 1 min at 72° C. The products of PCR were analyzedon 1% agarose gels. Then 5 μL of this mixture was used for the secondround of amplification under the same conditions, except the extensiontime was increased to 3 min.

The RT-PCR products produced in all dilutions of the standard HEV fecesin the range from 10⁻¹ to 10⁻⁵ (FIG. 9) were separated on a 2% agarosegel and were detected by ethiduim bromide staining of the gel. Adecrease in the amount of the specific PCR product at higher dilutionswas observed and the highest dilution of the 10% fecal suspension inwhich the HEV genome was detected was 10⁻⁵. Therefore, taking intoaccount the dilution factor, the HEV genome titer was approximately106.7 per gram of feces.

In addition, only those dilutions that were shown by RT-PCR to containthe HEV genome were infectious for cynomolgus monkeys. Therefore, theinfectivity titer of the standard fecal suspension and its genome titeras detected by RT-PCR were approximately the same. A similar correlationbetween RT-PCR and infectivity titer was found for one strain ofhepatitis C virus [Cristiano et al., (1991) Henatology, 14:51-55; Farciet al., (1991) N. Engl. J. Med., 25:98-104; Bukh et al., (1992); Proc.Natl. Acad. Sci U.S.A., 89:187-191)

EXAMPLE 12 Active Immunization Using the ORF-2 Protein as a Vaccine andPassive Immunization With Anti-HEV Positive Convalescent Plasma

Cynomolgus monkeys (Macaca fascicularis) that were HEV antibody negative(<1:10) in an ELISA based on the 55 kDa ORF-2 protein were individuallyhoused under BL-2 biohazard containment and a suspension (in fetalbovine serum) of feces containing the Pakistani HEV strain SAR-55,diluted to contain 10,000 or 1,000 CID₅₀, was used for intravenousinoculation of animals.

For active immunization studies, baculovirus recombinant-expressed 55kDa ORF-2 protein was purified from 5×10⁸ SF-9 cells harvested 7 dayspost-inoculation as described in Example 10. Three mg of the purified 55kDa protein were precipitated with alum and eight cynomolgus monkeyswere immunized by intramuscular injection with 0.5 ml of vaccinecontaining 50 μg of the alum-precipitated 55 kDa protein. Four monkeysreceived a single dose and four monkeys received two doses separated byfour weeks. Primates were challenged intravenously with 1,000-10,000CID₅₀ of HEV four weeks after the last immunization.

Four cynomolgus monkeys served as controls in the active immunizationstudies. Cyno-412 and 413 received one dose of placebo (0.5 ml ofphosphate buffered saline) and cyno-397 and 849 received two doses ofplacebo. The control animals were challenged with 1,000-10,000 CID₅₀ ofHEV.

For passive immunity studies, cyno-384 was infected with 0.5 ml of a 10%pooled stool suspension containing two Chinese HEV isolates, KS1-1987and KS2-1987 and plasma was repeatedly collected from the animal duringconvalesence. (Yin et al. (1993) J. Med. Virol., 41:230-241;).Approximately 1% of the blood of cyno-396 and cyno-399 and 10% of theblood of cyno-401 and cyno-402 was replaced with convalescent plasmafrom cyno-384 having an HEV antibody titer of 1:10,000. Animals werechallenged with 1000 CID₅₀ of HEV two days after infusion of the plasma.As a control, 10% of the blood of cyno-405 was replaced with anti-HEVnegative plasma obtained from cyno-384 prior to infection with HEV.Cyno-405 was then challenged with 1000 CID₅₀ of HEV.

For both the passive and active immunization studies, percutaneousneedle biopsies of the liver and samples of serum and feces werecollected prior to inoculation and weekly for 15 weeks afterinoculation. Sera were assayed for levels of alanine amino transferase(ALT) with commercially available tests (Metpath Inc., Rockville, Md.)and biochemical evidence of hepatitis was defined as a two-fold orgreater increase in ALT. Liver biopsies were examined under code and theanti-HEV ELISA utilized was described in Example 10. RNA extraction andRT-PCR were performed as in Example 11 except that RNA from 100 μl ofserum or from 100 μl of 10% fecal suspension was extracted with TRIzolReagent (Gibco BRL, Gaithersburg, Md.) according to the manufacturer'sprotocol. For quantification, PCR positive serial sera or feces fromeach animal were combined and serially diluted in ten-fold increments incalf serum. One hundred μl of each dilution were used for RNA extractionand RT-PCR as described earlier in this Example. The PCR protocol usedin this study could detect as few as 10 CID₅₀ of HEV per ml of serum andas few as 100 CID₅₀ per gram of feces.

Peak ALT values of weekly serum samples for 5 weeks prior to inoculationand for 15 weeks post-inoculation were expressed as ratios (post/pre)for each animal. The geometric mean of the ratios from the control groupof animals was compared with that from the passively or activelyimmunized animals using the Simes test (Simes, R. J. (1986) Biometrika,73:751-754).

The durations of viremia and virus shedding in feces and the HEV genometiters in the control group of animals were compared with those inpassively or actively immunized animals using the Wilcoxon test[Noether, G. (1967) in Elements of nonparametric statistics (John Wiley& Sons Inc., New York), pp. 31-36.]. The same test was used to comparethe above parameters between passively and actively immunized animals.

For statistical analysis, serum samples that had <10 HEV genomes in 1 mlof serum were assigned a titer of 1:1 and fecal samples that had <100HEV genomes in 1 g of feces were assigned a titer of 1:10.

RESULTS

Course of hepatitis E infection in nonimmunized animals.

In 3 of 5 nonimmunized animals that were challenged with HEV,biochemical evidence of hepatitis was documented by at least a two-foldincrease in serum ALT values. In two animals, significant increases inALT activity were not found. However, histopathological data documentedhepatitis in all 5 animals as shown in Table 6.

TABLE 6 Histopathological, biochemical, serological, and virologicalprofiles of vaccinated and control animals challenged with HEV. Anti-HEVCumulative HEV HEV genome positive score of Peak ALT antibody serumfeces Animal # plasma histopathology value in U/L (week) titer at theweek mean week mean and (%) or 55 kDA (number of pre- post- time ofdetected log₁₀ titer detected log₁₀ titer category protein (μg) weeksdetected)*. inoculation inoculation challenge (duration) per ml(duration) per gram control 405 0 10+ (8) 67 (0) 143 (9) <1:10 1-11 (11)3 1-11 (11) 5.7 412 0 2+ (1) 34 (0) 45 (3) <1:10 1-4 (4) 3 2-5 (4) 7 4130 4+ (4) 44 (0) 261 (6) <1:10 2-7 (6) 4.7 1-7 (7) 7 849 0 1+ (1) 79 (−2)133 (2) <1:10 1-4 (4) 3.7 1-4 (4) 7 397 0 3+ (3) 52 (−3) 139 (7) <1:102-6 (5) 4.7 1-7 (7) 7 passive IP^(†) 396 1% 1+ (1)^(‡) 33 (0) 53 (6)1:40 3-5 (3) 4 1-6 (6) 5.7 399 1% 0 (0) 69 (0) 63 (11) 1:40 2-4 (3) 31-4 (4) 4 401 10% 0 (0) 55 (0) 45 (5) 1:200 3 (1) 3.6 1-3 (3) 5.7 40210% 0 (0) 59 (0) 35 (2) 1:200 4-6 (3) 1 2-6 (5) 5.7 20 active IP^(†) 00350 μg 0 (0) 34 (−3) 50 (6) 1:10,000 0 <1 2-4 (3) 3 009 50 μg 0 (0) 34(−2) 38 (6) 1:1,000 0 <1 0 <2 013^(§) 50 μg 0 (0) 44 (−3) 36 (7) 1:100 0<1 1-2 (2) 3 414 50 μg 0 (0) 65 (0) 73 (8) 1:1,000 0 <1 2 (1) 2 398 2 ×50 μg 0 (0) 31 (0) 41 (2) 1:10,000 0 <1 0 <2 407 2 × 50 μg 0 (0) 150 (0)213 (4) 1:10,000 0 <1 0 <2 *Necro-inflammatory changes in the liver wererated as 1+, 2+, 3+, 4+ and the weekly scores were summed.^(†)Immunoprophylaxis ^(‡)Necro-inflammatory changes rated 1+ weredetected during two weeks in cyno-396, however, they were consistentwith viral hepatitis only during one week. ^(§)Cyno 013 died 9 weeksafter challenge.

Necro-inflammatory changes ranged between 1+ and 2+ on a scale of 1+ to4+ and were temporally associated with elevations of ALT activities inthose animals with such elevations.

All control animals seroconverted to HEV 3-5 weeks post-challenge anddeveloped maximum HEV antibody titers ranging from 1:1,000 to 1:32,000.There was a good correlation between the severity of infection,hepatitis, and the level of anti-HEV response. Cyno-405, which had thehighest cumulative score for hepatitis, also had the longest period ofviremia and viral excretion and the highest level of anti-HEV (Table 6).The duration of viral shedding in feces was the same as, or longer than,that of the viremia. For all of the control animals, titers of the HEVgenome in serum were lower (10⁻³-10^(−4.7)) than the titers in feces(10^(−5.7)-10⁻⁷). In all five of these animals, viremia and virusshedding in feces were detected for 4-11 weeks and for an average of 4.2weeks after seroconversion (range 2-9 weeks).

Passive immunization. Cyno-396 and 399, which had approximately 1% oftheir blood replaced with anti-HEV positive convalescent plasma, had anHEV antibody titer of 1:40 when it was determined two dayspost-transfusion (at the time of challenge) (Table 6). A two-fold fallin HEV antibody titer was observed in both animals 1 weekpost-transfusion and HEV antibodies fell below the detectable level(<1:10) by 2 weeks post-transfusion. Anti-HEV was again detected 5 weekspost-challenge in cyno-396 and 4 weeks post-challenge in cyno-399,indicating that infection with HEV had occurred. The maximum HEVantibody titer (1:8,000) was reached 9-10 weeks post-challenge. Neithercynomolgus monkey demonstrated a significant elevation of ALT activityafter challenge. However, histologic evidence of hepatitis was detectedin cyno-396 and the HEV genome was detected in serum and feces from bothanimals (Table 6).

Cyno-401 and 402 had approximately 10% of their blood replaced withconvalescent plasma. Two days post-transfusion, at the time ofchallenge, the HEV antibody titer in both cynomolgus monkeys was 1:200(Table 7).

TABLE 7 HEV antibody profiles in control and immunized cynomolgusmonkeys. HEV antobody HEV antibody HEV antibody max. titer max. titertiter Passively titer at max. titer Actively (week (week max. titerControl (week first max. titer immunized the time of (week afterimmunized after 1st after 2nd (week after animals detected) (week)animals challenge challenge) animals immunization) immunizationchallenge) cyno-405 1:80  1:32,000 cyno-396 1:40  1:8,000 cyno-0031:10,000 1:10,000 (3) (9) (10) (3) (5) cyno-412 1:100 1:10,000 cyno-3991:40  1:8,000 cyno-009 1:10,000 1:10,000 (5) (7)  (9) (3) (1) cyno-4131:100 1:10,000 cyno-401 1:200 1:4,000 cyno-013 1:100   1:10,000 (5) (7) (6) (2) (3) cyno-849 1:100 1:1,000  cyno-402 1:200 1:80   cyno-4141:1,000 1:1,000  (3) (5) (12) (3) (0) cyno-397 1:100 1:10,000 cyno-3981:1,000 1:10,000 1:10,000 (3) (7) (3) (5) (0) cyno-407 1:1,000 1:10,0001:10,000 (4) (5) (0)

Anti-HEV was detected continuously in both animals during the 15 weeksafter challenge and reached a maximum titer of 1:4,000 in cyno-401 butonly 1:80 in cyno-402. Biochemical and histologic analyses did notreveal hepatitis in either animal. However, in both animals, HEV viremiaand fecal shedding of virus were observed indicating that infection hadoccurred (Table 6). Thus, passive immunoprophylaxis that achieved ahigher titer of antibody protected cynomolgus monkeys against hepatitisafter challenge with HEV.

Active immunization. Four primates immunized with one 50 μg dose of the55 kDa protein developed antibody to the recombinant protein ranging intiter from 1:100 to 1:10,000 (Table 7). One (cyno 013) died of ananesthesia accident 9 weeks after challenge and is included in theanalyses (Table 6). The four animals that received two doses of theantigen developed HEV antibodies with titers of 1:10,000. Two of thefour monkeys died following intravenous challenge with HEV. This mayhave also been the result of an anesthesia accident but the exactetiology could not be determined. These two monkeys were deleted fromfurther analyses. None of the 6 remaining animals developed abnormal ALTlevels or histologic evidence of hepatitis following challenge (Table6). Cynomolgus monkeys immunized with either 1 or 2 doses of the 55 kDaprotein did not develop viremia. However, 3 of 4 animals that receivedone dose of the immunogen excreted virus in their feces. In contrast,virus shedding was not observed in either of the two challenged animalsthat had received two doses of the vaccine.

Most of the actively immunized animals developed higher HEV antibodytiters than did passively immunized animals. However, cyno-013 had anHEV antibody titer of 1:100 at the time of challenge, compared with atiter of 1:200 in two animals immunized passively with anti-HEV plasma.Cyno-013, however, demonstrated greater protection against HEV infectionthan the passively immunized animals. Cyno-009, which had an HEVantibody titer of 1:1,000 at the time of challenge, was completelyprotected against hepatitis and HEV infection (Table 6). In contrast,cyno-003 was infected and shed HEV in feces, even though it had an HEVantibody titer of 1:10,000 at the time of challenge. However, neitherhepatitis nor viremia was detected in this animal or in other cynomolgusmonkeys that received one dose of immunogen and had HEV antibody titersof 1:10,000 or greater.

Comparison of course of HEV infection in control and immunized animals.

As measured by histopathology, all immunized animals, with the exceptionof one of the passively immunized monkeys, were protected againsthepatitis after intravenous challenge with HEV. Comparison of meanvalues for severity of hepatitis and level of viral replication betweenthe control group and the passively and actively immunized animalsindicated that, in general, the severity of infection was inverselyrelated to the HEV antibody titer at the time of challenge anddiminished in the order: unimmunized>passive immunization (1%)>passiveimmunization (10%)>active immunization (1 dose)>active immunization (2doses) (Tables 6,8). However, the number of animals in each of the twosubgroups of passively and actively immunized animals was not sufficientto permit statistical analysis. Therefore, statistical analysis wasperformed for combined passively immunized and combined activelyimmunized groups respectively in comparison with the combined controlgroups. The results of this analysis are presented in Table 8.

TABLE 8 Summary of mean values of HEV infection in control and immunizedanimals.

*Geometric mean ^(†)Passive and active immunoprophylaxis α - P < 0.01β - P < 0.05 γ - not significant

and they show that the histopathology scores and duration of histologicchanges in the control group were statistically different from those ofpassively or actively immunized animals. The higherpost-/pre-inoculation ratios of peak ALT values in the control groupwere statistically significant when compared with those of the passivelyor actively immunized animals, indicating protection against biochemicalmanifestations of hepatitis in both groups of immunized animals. Theduration of viremia and the titer of HEV in the feces were significantlylower in both groups of immunized animals than in the control group.Differences in the duration of virus shedding and titer of HEV in theserum, however, were not statistically different between the controlgroup and the passively immunized group, although these parameters weresignificantly different when the control group was compared with theactively immunized group. Significant differences were also foundbetween passively and actively immunized groups of animals for durationof viremia and fecal shedding as well as for HEV titers.

In sum, the results presented in Tables 6-8 show that both passively andactively acquired HEV antibodies protected cynomolgus monkeys againsthepatitis following challenge with virulent HEV. Although all 5nonimmunized cynomolgus monkeys developed histologic evidence ofhepatitis when challenged with 1,000-10,000 CID₅₀ of SAR-55, bothanimals with passively acquired antibody titers of 1:200 were protectedfrom hepatitis and one of two animals with an antibody titer as low as1:40 also did not develop hepatitis.

However, it should be noted that actively immunized animals demonstratedcomplete protection against hepatitis and more effective resistance toHEV infection than did passively immunized animals. For example, incontrast to results obtained from the passively immunized animals,viremia was not detected in actively immunized animals after challengewith HEV. An HEV antibody titer as high as 1:10,000 could be achieved incynomolgus monkeys after one or two immunizations with the recombinant55 kDa protein. Although one monkey (013) developed a titer of 1:100after active immunization, this level still prevented hepatitis andviremia.

The active immunization studies also demonstrated that while a singledose of vaccine prevented HEV viremia, viral shedding in feces was stilldetected. However, two doses of vaccine were observed to prevent allsigns of hepatitis and HEV infection. These results thus suggest that asingle dose of vaccine administered, for example, to individuals beforeforeign travel would protect them from hepatitis E in high riskenvironments.

Finally, it is noted that the results presented are very similar toresults reported previously for passive and active immunoprophylaxis ofnonhuman primates against hepatitis A: passive immunoprophylaxisprevented hepatitis but not infection whereas vaccination prevented notonly hepatitis but infection with HAV as well (Purcell, R. H. et al.(1992) Vaccine, 10:5148-5149). It is of interest that the study ofimmunoprophylaxis for HEV presented herein parallels the previous studyof immunoprophylaxis against HAV, both in determination of the titer ofantibody that protected (<1:100) and in outcome following intravenouschallenge with virulent virus. Since other studies have demonstratedefficacy of comparable titers of passively and actively acquiredanti-HAV in humans and have confirmed the predictive value of studies ofprimates in hepatitis research (Stapleton, J., et al. (1985)Gastroenterology 89:637-642; Innis, B. L., et al. (1992) Vaccine, 10:S159), it is therefore highly likely that these results in cynomolgusmonkeys will be predictive of protection in humans.

EXAMPLE 13 Direct Expression in Yeast of Complete ORF-2 Protein andLower Molecular Weight Fragments

Four cDNA ORF-2 fragments coding for:

1. complete ORF-2 protein (aa 1-660, MW 70979), fragment 1778-1703.(where the fragment numbers refer to the primer numbers given below)

2. ORF-2 protein starting from 34th aa (aa 34-660, MW 67206), fragment1779-1703.

3. ORF-2 protein starting from 96th aa (aa 96-660, MW 60782), fragment1780-1703.

4. ORF-2 protein starting from 124th aa (aa 124-660, MW 58050), fragment1781-1703.

were obtained using PCR by using plasmid P63-2 as template and thesynthetic oligonucleotides shown below:

SEQ ID NO.:103 (reverse primer #1703)

GCACAACCTAGGTTACTATAACTCCCGAGTTTTACC, SEQ ID NO.:104

(direct primer #1778) GGGTTCCCTAGGATGCGCCCTCGGCCTATTTTG,

SEQ ID NO.:105 (direct primer #1779)

CGTGGGCCTAGGAGCGGCGGTTCCGGCGGTGGT, SEQ ID NO.:106 (direct primer #1780)GCTTGGCCTAGGCAGGCCCAGCGCCCCGCCGCT and SEQ ID NO.:107 (direct primer#1781)

CCGCCACCTAGGGATGTTGACTCCCGCGGCGCC.

All sequences shown in SEQ ID NOs: 103-107 contain artificial sequenceCCTAGG at their 5′ ends preceded by 4 nucleotides. The artificialsequence was a recognition site for Avr II (Bln I) restriction enzyme.Synthesized PCR fragments were cleaved with BlnI and cloned in the AvrIIsite of pPIC9 vector (FIG. 10) (Invitrogen). Correct orientation of thefragments was confirmed by restriction analysis, using asymmetric EcoRIsite present in ORF-2 sequences and in the vector. Purified recombinantplasmids pPIC9-1778 (containing fragment 1778-1703); pPIC9-1779(containing fragment 1779-1703); pPIC9-1780 (containing fragment1780-1703) and pPIC9-1781 (containing fragment 1781-1730) were used fortransformation of yeast spheroplast (Picha strain) according toInvitrogen protocol. Screening of recombinant clones and analysis ofexpression were performed using the same protocol. These expressedproteins may be used as immunogens in vaccines and as antigens inimmunoassays as described in the present application. Finally, those ofskill in the art would recognize that the vector and strain of yeastused in the above example could be replaced by other vectors (e.g.pHIL-F1; Invitrogen) or strains of yeast (e.g. SaccharomycesCerevisiae).

107 1693 AMINO ACID RESIDUES AMINO ACID UNKNOWN UNKNOWN 1 Met Glu AlaHis Gln Phe Ile Lys Ala Pro Gly Ile Thr Thr Ala 1 5 10 15 Ile Glu GlnAla Ala Leu Ala Ala Ala Asn Ser Ala Leu Ala Asn 20 25 30 Ala Val Val ValArg Pro Phe Leu Ser His Gln Gln Ile Glu Ile 35 40 45 Leu Ile Asn Leu MetGln Pro Arg Gln Leu Val Phe Arg Pro Glu 50 55 60 Val Phe Trp Asn His ProIle Gln Arg Val Ile His Asn Glu Leu 65 70 75 Glu Leu Tyr Cys Arg Ala ArgSer Gly Arg Cys Leu Glu Ile Gly 80 85 90 Ala His Pro Arg Ser Ile Asn AspAsn Pro Asn Val Val His Arg 95 100 105 Cys Phe Leu Arg Pro Ala Gly ArgAsp Val Gln Arg Trp Tyr Thr 110 115 120 Ala Pro Thr Arg Gly Pro Ala AlaAsn Cys Arg Arg Ser Ala Leu 125 130 135 Arg Gly Leu Pro Ala Ala Asp ArgThr Tyr Cys Phe Asp Gly Phe 140 145 150 Ser Gly Cys Asn Phe Pro Ala GluThr Gly Ile Ala Leu Tyr Ser 155 160 165 Leu His Asp Met Ser Pro Ser AspVal Ala Glu Ala Met Phe Arg 170 175 180 His Gly Met Thr Arg Leu Tyr AlaAla Leu His Leu Pro Pro Glu 185 190 195 Val Leu Leu Pro Pro Gly Thr TyrArg Thr Ala Ser Tyr Leu Leu 200 205 210 Ile His Asp Gly Arg Arg Val ValVal Thr Tyr Glu Gly Asp Thr 215 220 225 Ser Ala Gly Tyr Asn His Asp ValSer Asn Leu Arg Ser Trp Ile 230 235 240 Arg Thr Thr Lys Val Thr Gly AspHis Pro Leu Val Ile Glu Arg 245 250 255 Val Arg Ala Ile Gly Cys His PheVal Leu Leu Leu Thr Ala Ala 260 265 270 Pro Glu Pro Ser Pro Met Pro TyrVal Pro Tyr Pro Arg Ser Thr 275 280 285 Glu Val Tyr Val Arg Ser Ile PheGly Pro Gly Gly Thr Pro Ser 290 295 300 Leu Phe Pro Thr Ser Cys Ser ThrLys Ser Thr Phe His Ala Val 305 310 315 Pro Ala His Ile Trp Asp Arg LeuMet Leu Phe Gly Ala Thr Leu 320 325 330 Asp Asp Gln Ala Phe Cys Cys SerArg Leu Met Thr Tyr Leu Arg 335 340 345 Gly Ile Ser Tyr Lys Val Thr ValGly Thr Leu Val Ala Asn Glu 350 355 360 Gly Trp Asn Ala Ser Glu Asp AlaLeu Thr Ala Val Ile Thr Ala 365 370 375 Ala Tyr Leu Thr Ile Cys His GlnArg Tyr Leu Arg Thr Gln Ala 380 385 390 Ile Ser Lys Gly Met Arg Arg LeuGlu Arg Glu His Ala Gln Lys 395 400 405 Phe Ile Thr Arg Leu Tyr Ser TrpLeu Phe Glu Lys Ser Gly Arg 410 415 420 Asp Tyr Ile Pro Gly Arg Gln LeuGlu Phe Tyr Ala Gln Cys Arg 425 430 435 Arg Trp Leu Ser Ala Gly Phe HisLeu Asp Pro Arg Val Leu Val 440 445 450 Phe Asp Glu Ser Ala Pro Cys HisCys Arg Thr Ala Ile Arg Lys 455 460 465 Ala Val Ser Lys Phe Cys Cys PheMet Lys Trp Leu Gly Gln Glu 470 475 480 Cys Thr Cys Phe Leu Gln Pro AlaGlu Gly Val Val Gly Asp Gln 485 490 495 Gly His Asp Asn Glu Ala Tyr GluGly Ser Asp Val Asp Pro Ala 500 505 510 Glu Ser Ala Ile Ser Asp Ile SerGly Ser Tyr Val Val Pro Gly 515 520 525 Thr Ala Leu Gln Pro Leu Tyr GlnAla Leu Asp Leu Pro Ala Glu 530 535 540 Ile Val Ala Arg Ala Gly Arg LeuThr Ala Thr Val Lys Val Ser 545 550 555 Gln Val Asp Gly Arg Ile Asp CysGlu Thr Leu Leu Gly Asn Lys 560 565 570 Thr Phe Arg Thr Ser Phe Val AspGly Ala Val Leu Glu Thr Asn 575 580 585 Gly Pro Glu Arg His Asn Leu SerPhe Asp Ala Ser Gln Ser Thr 590 595 600 Met Ala Ala Gly Pro Phe Ser LeuThr Tyr Ala Ala Ser Ala Ala 605 610 615 Gly Leu Glu Val Arg Tyr Val AlaAla Gly Leu Asp His Arg Ala 620 625 630 Val Phe Ala Pro Gly Val Ser ProArg Ser Ala Pro Gly Glu Val 635 640 645 Thr Ala Phe Cys Ser Ala Leu TyrArg Phe Asn Arg Glu Ala Gln 650 655 660 Arg Leu Ser Leu Thr Gly Asn PheTrp Phe His Pro Glu Gly Leu 665 670 675 Leu Gly Pro Phe Ala Pro Phe SerPro Gly His Val Trp Glu Ser 680 685 690 Ala Asn Pro Phe Cys Gly Glu SerThr Leu Tyr Thr Arg Thr Trp 695 700 705 Ser Glu Val Asp Ala Val Pro SerPro Ala Gln Pro Asp Leu Gly 710 715 720 Phe Thr Ser Glu Pro Ser Ile ProSer Arg Ala Ala Thr Pro Thr 725 730 735 Pro Ala Ala Pro Leu Pro Pro ProAla Pro Asp Pro Ser Pro Thr 740 745 750 Leu Ser Ala Pro Ala Arg Gly GluPro Ala Pro Gly Ala Thr Ala 755 760 765 Arg Ala Pro Ala Ile Thr His GlnThr Ala Arg His Arg Arg Leu 770 775 780 Leu Phe Thr Tyr Pro Asp Gly SerLys Val Phe Ala Gly Ser Leu 785 790 795 Phe Glu Ser Thr Cys Thr Trp LeuVal Asn Ala Ser Asn Val Asp 800 805 810 His Arg Pro Gly Gly Gly Leu CysHis Ala Phe Tyr Gln Arg Tyr 815 820 825 Pro Ala Ser Phe Asp Ala Ala SerPhe Val Met Arg Asp Gly Ala 830 835 840 Ala Ala Tyr Thr Leu Thr Pro ArgPro Ile Ile His Ala Val Ala 845 850 855 Pro Asp Tyr Arg Leu Glu His AsnPro Lys Arg Leu Glu Ala Ala 860 865 870 Tyr Arg Glu Thr Cys Ser Arg LeuGly Thr Ala Ala Tyr Pro Leu 875 880 885 Leu Gly Thr Gly Ile Tyr Gln ValPro Ile Gly Pro Ser Phe Asp 890 895 900 Ala Trp Glu Arg Asn His Arg ProGly Asp Glu Leu Tyr Leu Pro 905 910 915 Glu Leu Ala Ala Arg Trp Phe GluAla Asn Arg Pro Thr Cys Pro 920 925 930 Thr Leu Thr Ile Thr Glu Asp ValAla Arg Thr Ala Asn Leu Ala 935 940 945 Ile Glu Leu Asp Ser Ala Thr AspVal Gly Arg Ala Cys Ala Gly 950 955 960 Cys Arg Val Thr Pro Gly Val ValGln Tyr Gln Phe Thr Ala Gly 965 970 975 Val Pro Gly Ser Gly Lys Ser ArgSer Ile Thr Gln Ala Asp Val 980 985 990 Asp Val Val Val Val Pro Thr ArgGlu Leu Arg Asn Ala Trp Arg 995 1000 1005 Arg Arg Gly Phe Ala Ala PheThr Pro His Thr Ala Ala Arg Val 1010 1015 1020 Thr Gln Gly Arg Arg ValVal Ile Asp Glu Ala Pro Ser Leu Pro 1025 1030 1035 Pro His Leu Leu LeuLeu His Met Gln Arg Ala Ala Thr Val His 1040 1045 1050 Leu Leu Gly AspPro Asn Gln Ile Pro Ala Ile Asp Phe Glu His 1055 1060 1065 Ala Gly LeuVal Pro Ala Ile Arg Pro Asp Leu Ala Pro Thr Ser 1070 1075 1080 Trp TrpHis Val Thr His Arg Cys Pro Ala Asp Val Cys Glu Leu 1085 1090 1095 IleArg Gly Ala Tyr Pro Met Ile Gln Thr Thr Ser Arg Val Leu 1100 1105 1110Arg Ser Leu Phe Trp Gly Glu Pro Ala Val Gly Gln Lys Leu Val 1115 11201125 Phe Thr Gln Ala Ala Lys Ala Ala Asn Pro Gly Ser Val Thr Val 11301135 1140 His Glu Ala Gln Gly Ala Thr Tyr Thr Glu Thr Thr Ile Ile Ala1145 1150 1155 Thr Ala Asp Ala Arg Gly Leu Ile Gln Ser Ser Arg Ala HisAla 1160 1165 1170 Ile Val Ala Leu Thr Arg His Thr Glu Lys Cys Val IleIle Asp 1175 1180 1185 Ala Pro Gly Leu Leu Arg Glu Val Gly Ile Ser AspAla Ile Val 1190 1195 1200 Asn Asn Phe Phe Leu Ala Gly Gly Glu Ile GlyHis Gln Arg Pro 1205 1210 1215 Ser Val Ile Pro Arg Gly Asn Pro Asp AlaAsn Val Asp Thr Leu 1220 1225 1230 Ala Ala Phe Pro Pro Ser Cys Glu IleSer Ala Phe His Glu Leu 1235 1240 1245 Ala Glu Glu Leu Gly His Arg ProAla Pro Val Ala Ala Val Leu 1250 1255 1260 Pro Pro Cys Pro Glu Leu GluGln Gly Leu Leu Tyr Leu Pro Gln 1265 1270 1275 Glu Leu Thr Thr Cys AspSer Val Val Thr Phe Glu Leu Thr Asp 1280 1285 1290 Ile Val His Cys ArgMet Ala Ala Pro Ser Gln Arg Lys Ala Val 1295 1300 1305 Leu Ser Thr LeuVal Gly Arg Tyr Gly Arg Arg Thr Lys Leu Tyr 1310 1315 1320 Asn Ala SerHis Ser Asp Val Arg Asp Ser Leu Ala Arg Phe Ile 1325 1330 1335 Pro AlaIle Gly Pro Val Gln Val Thr Thr Cys Glu Leu Tyr Glu 1340 1345 1350 LeuGlu Glu Ala Met Val Glu Lys Gly Gln Asp Gly Ser Ala Val 1355 1360 1365Leu Glu Leu Asp Leu Cys Ser Arg Asp Val Ser Arg Ile Thr Phe 1370 13751380 Phe Gln Lys Asp Cys Asn Lys Phe Thr Thr Gly Glu Thr Ile Ala 13851390 1395 His Gly Lys Val Gly Gln Gly Ile Ser Ala Trp Ser Lys Thr Phe1400 1405 1410 Cys Ala Leu Phe Gly Pro Trp Phe Arg Ala Ile Glu Lys AlaIle 1415 1420 1425 Leu Ala Leu Leu Pro Gln Gly Val Phe Tyr Gly Asp AlaPhe Asp 1430 1435 1440 Asp Thr Val Phe Ser Ala Ala Val Ala Ala Ala LysAla Ser Met 1445 1450 1455 Val Phe Glu Asn Asp Phe Ser Glu Phe Asp SerThr Gln Asn Asn 1460 1465 1470 Phe Ser Leu Gly Leu Glu Cys Ala Ile MetGlu Glu Cys Gly Met 1475 1480 1485 Pro Gln Trp Leu Ile Arg Leu Tyr HisLeu Ile Arg Ser Ala Trp 1490 1495 1500 Ile Leu Gln Ala Pro Lys Glu SerLeu Arg Gly Phe Trp Lys Lys 1505 1510 1515 His Ser Gly Glu Pro Gly ThrLeu Leu Trp Asn Thr Val Trp Asn 1520 1525 1530 Met Ala Val Ile Thr HisCys Tyr Asp Phe Arg Asp Leu Gln Val 1535 1540 1545 Ala Ala Phe Lys GlyAsp Asp Ser Ile Val Leu Cys Ser Glu Tyr 1550 1555 1560 Arg Gln Ser ProGly Ala Ala Val Leu Ile Ala Gly Cys Gly Leu 1565 1570 1575 Lys Leu LysVal Asp Phe Arg Pro Ile Gly Leu Tyr Ala Gly Val 1580 1585 1590 Val ValAla Pro Gly Leu Gly Ala Leu Pro Asp Val Val Arg Phe 1595 1600 1605 AlaGly Arg Leu Thr Glu Lys Asn Trp Gly Pro Gly Pro Glu Arg 1610 1615 1620Ala Glu Gln Leu Arg Leu Ala Val Ser Asp Phe Leu Arg Lys Leu 1625 16301635 Thr Asn Val Ala Gln Met Cys Val Asp Val Val Ser Arg Val Tyr 16401645 1650 Gly Val Ser Pro Gly Leu Val His Asn Leu Ile Glu Met Leu Gln1655 1660 1665 Ala Val Ala Asp Gly Lys Ala His Phe Thr Glu Ser Val LysPro 1670 1675 1680 Val Leu Asp Leu Thr Asn Ser Ile Leu Cys Arg Val Glu1685 1690 660 amino acid residues amino acid unknown unknown 2 Met ArgPro Arg Pro Ile Leu Leu Leu Leu Leu Met Phe Leu Pro 1 5 10 15 Met LeuPro Ala Pro Pro Pro Gly Gln Pro Ser Gly Arg Arg Arg 20 25 30 Gly Arg ArgSer Gly Gly Ser Gly Gly Gly Phe Trp Gly Asp Arg 35 40 45 Val Asp Ser GlnPro Phe Ala Ile Pro Tyr Ile His Pro Thr Asn 50 55 60 Pro Phe Ala Pro AspVal Thr Ala Ala Ala Gly Ala Gly Pro Arg 65 70 75 Val Arg Gln Pro Ala ArgPro Leu Gly Ser Ala Trp Arg Asp Gln 80 85 90 Ala Gln Arg Pro Ala Ala AlaSer Arg Arg Arg Pro Thr Thr Ala 95 100 105 Gly Ala Ala Pro Leu Thr AlaVal Ala Pro Ala His Asp Thr Pro 110 115 120 Pro Val Pro Asp Val Asp SerArg Gly Ala Ile Leu Arg Arg Gln 125 130 135 Tyr Asn Leu Ser Thr Ser ProLeu Thr Ser Ser Val Ala Thr Gly 140 145 150 Thr Asn Leu Val Leu Tyr AlaAla Pro Leu Ser Pro Leu Leu Pro 155 160 165 Leu Gln Asp Gly Thr Asn ThrHis Ile Met Ala Thr Glu Ala Ser 170 175 180 Asn Tyr Ala Gln Tyr Arg ValAla Arg Ala Thr Ile Arg Tyr Arg 185 190 195 Pro Leu Val Pro Asn Ala ValGly Gly Tyr Ala Ile Ser Ile Ser 200 205 210 Phe Tyr Pro Gln Thr Thr ThrThr Pro Thr Ser Val Asp Met Asn 215 220 225 Ser Ile Thr Ser Thr Asp ValArg Ile Leu Val Gln Pro Gly Ile 230 235 240 Ala Ser Glu Leu Val Ile ProSer Glu Arg Leu His Tyr Arg Asn 245 250 255 Gln Gly Trp Arg Ser Val GluThr Ser Gly Val Ala Glu Glu Glu 260 265 270 Ala Thr Ser Gly Leu Val MetLeu Cys Ile His Gly Ser Pro Val 275 280 285 Asn Ser Tyr Thr Asn Thr ProTyr Thr Gly Ala Leu Gly Leu Leu 290 295 300 Asp Phe Ala Leu Glu Leu GluPhe Arg Asn Leu Thr Pro Gly Asn 305 310 315 Thr Asn Thr Arg Val Ser ArgTyr Ser Ser Thr Ala Arg His Arg 320 325 330 Leu Arg Arg Gly Ala Asp GlyThr Ala Glu Leu Thr Thr Thr Ala 335 340 345 Ala Thr Arg Phe Met Lys AspLeu Tyr Phe Thr Ser Thr Asn Gly 350 355 360 Val Gly Glu Ile Gly Arg GlyIle Ala Leu Thr Leu Phe Asn Leu 365 370 375 Ala Asp Thr Leu Leu Gly GlyLeu Pro Thr Glu Leu Ile Ser Ser 380 385 390 Ala Gly Gly Gln Leu Phe TyrSer Arg Pro Val Val Ser Ala Asn 395 400 405 Gly Glu Pro Thr Val Lys LeuTyr Thr Ser Val Glu Asn Ala Gln 410 415 420 Gln Asp Lys Gly Ile Ala IlePro His Asp Ile Asp Leu Gly Glu 425 430 435 Ser Arg Val Val Ile Gln AspTyr Asp Asn Gln His Glu Gln Asp 440 445 450 Arg Pro Thr Pro Ser Pro AlaPro Ser Arg Pro Phe Ser Val Leu 455 460 465 Arg Ala Asn Asp Val Leu TrpLeu Ser Leu Thr Ala Ala Glu Tyr 470 475 480 Asp Gln Ser Thr Tyr Gly SerSer Thr Gly Pro Val Tyr Val Ser 485 490 495 Asp Ser Val Thr Leu Val AsnVal Ala Thr Gly Ala Gln Ala Val 500 505 510 Ala Arg Ser Leu Asp Trp ThrLys Val Thr Leu Asp Gly Arg Pro 515 520 525 Leu Ser Thr Ile Gln Gln TyrSer Lys Thr Phe Phe Val Leu Pro 530 535 540 Leu Arg Gly Lys Leu Ser PheTrp Glu Ala Gly Thr Thr Lys Ala 545 550 555 Gly Tyr Pro Tyr Asn Tyr AsnThr Thr Ala Ser Asp Gln Leu Leu 560 565 570 Val Glu Asn Ala Ala Gly HisArg Val Ala Ile Ser Thr Tyr Thr 575 580 585 Thr Ser Leu Gly Ala Gly ProVal Ser Ile Ser Ala Val Ala Val 590 595 600 Leu Ala Pro His Ser Val LeuAla Leu Leu Glu Asp Thr Met Asp 605 610 615 Tyr Pro Ala Arg Ala His ThrPhe Asp Asp Phe Cys Pro Glu Cys 620 625 630 Arg Pro Leu Gly Leu Gln GlyCys Ala Phe Gln Ser Thr Val Ala 635 640 645 Glu Leu Gln Arg Leu Lys MetLys Val Gly Lys Thr Arg Glu Leu 650 655 660 123 amino acid residuesamino acid unknown unknown 3 Met Asn Asn Met Ser Phe Ala Ala Pro Met GlySer Arg Pro Cys 1 5 10 15 Ala Leu Gly Leu Phe Cys Cys Cys Ser Ser CysPhe Cys Leu Cys 20 25 30 Cys Pro Arg His Arg Pro Val Ser Arg Leu Ala AlaVal Val Gly 35 40 45 Gly Ala Ala Ala Val Pro Ala Val Val Ser Gly Val ThrGly Leu 50 55 60 Ile Leu Ser Pro Ser Gln Ser Pro Ile Phe Ile Gln Pro ThrPro 65 70 75 Ser Pro Pro Met Ser Pro Leu Arg Pro Gly Leu Asp Leu Val Phe80 85 90 Ala Asn Pro Pro Asp His Ser Ala Pro Leu Gly Val Thr Arg Pro 95100 105 Ser Ala Pro Pro Leu Pro His Val Val Asp Leu Pro Gln Leu Gly 110115 120 Pro Arg Arg 7168 base pairs nucleic acid single linear 4AGGCAGACCA CATATGTGGT CGATGCCATG GAGGCCCATC AGTTTATCAA 50 GGCTCCTGGCATCACTACTG CTATTGAGCA GGCTGCTCTA GCAGCGGCCA 100 ACTCTGCCCT TGCGAATGCTGTGGTAGTTA GGCCTTTTCT CTCTCACCAG 150 CAGATTGAGA TCCTTATTAA CCTAATGCAACCTCGCCAGC TTGTTTTCCG 200 CCCCGAGGTT TTCTGGAACC ATCCCATCCA GCGTGTTATCCATAATGAGC 250 TGGAGCTTTA CTGTCGCGCC CGCTCCGGCC GCTGCCTCGA AATTGGTGCC300 CACCCCCGCT CAATAAATGA CAATCCTAAT GTGGTCCACC GTTGCTTCCT 350CCGTCCTGCC GGGCGTGATG TTCAGCGTTG GTATACTGCC CCTACCCGCG 400 GGCCGGCTGCTAATTGCCGG CGTTCCGCGC TGCGCGGGCT CCCCGCTGCT 450 GACCGCACTT ACTGCTTCGACGGGTTTTCT GGCTGTAACT TTCCCGCCGA 500 GACGGGCATC GCCCTCTATT CTCTCCATGATATGTCACCA TCTGATGTCG 550 CCGAGGCTAT GTTCCGCCAT GGTATGACGC GGCTTTACGCTGCCCTCCAC 600 CTCCCGCCTG AGGTCCTGTT GCCCCCTGGC ACATACCGCA CCGCGTCGTA650 CTTGCTGATC CATGACGGCA GGCGCGTTGT GGTGACGTAT GAGGGTGACA 700CTAGTGCTGG TTATAACCAC GATGTTTCCA ACCTGCGCTC CTGGATTAGA 750 ACCACTAAGGTTACCGGAGA CCACCCTCTC GTCATCGAGC GGGTTAGGGC 800 CATTGGCTGC CACTTTGTCCTTTTACTCAC GGCTGCTCCG GAGCCATCAC 850 CTATGCCCTA TGTCCCTTAC CCCCGGTCTACCGAGGTCTA TGTCCGATCG 900 ATCTTCGGCC CGGGTGGCAC CCCCTCCCTA TTTCCAACCTCATGCTCCAC 950 CAAGTCGACC TTCCATGCTG TCCCTGCCCA TATCTGGGAC CGTCTCATGT1000 TGTTCGGGGC CACCCTAGAT GACCAAGCCT TTTGCTGCTC CCGCCTAATG 1050ACTTACCTCC GCGGCATTAG CTACAAGGTT ACTGTGGGCA CCCTTGTTGC 1100 CAATGAAGGCTGGAACGCCT CTGAGGACGC TCTTACAGCT GTCATCACTG 1150 CCGCCTACCT TACCATCTGCCACCAGCGGT ACCTCCGCAC TCAGGCTATA 1200 TCTAAGGGGA TGCGTCGCCT GGAGCGGGAGCATGCTCAGA AGTTTATAAC 1250 ACGCCTCTAC AGTTGGCTCT TTGAGAAGTC CGGCCGTGATTATATCCCCG 1300 GCCGTCAGTT GGAGTTCTAC GCTCAGTGTA GGCGCTGGCT CTCGGCCGGC1350 TTTCATCTTG ACCCACGGGT GTTGGTTTTT GATGAGTCGG CCCCCTGCCA 1400CTGTAGGACT GCGATTCGTA AGGCGGTCTC AAAGTTTTGC TGCTTTATGA 1450 AGTGGCTGGGCCAGGAGTGC ACCTGTTTTC TACAACCTGC AGAAGGCGTC 1500 GTTGGCGACC AGGGCCATGACAACGAGGCC TATGAGGGGT CTGATGTTGA 1550 CCCTGCTGAA TCCGCTATTA GTGACATATCTGGGTCCTAC GTAGTCCCTG 1600 GCACTGCCCT CCAACCGCTT TACCAAGCCC TTGACCTCCCCGCTGAGATT 1650 GTGGCTCGTG CAGGCCGGCT GACCGCCACA GTAAAGGTCT CCCAGGTCGA1700 CGGGCGGATC GATTGTGAGA CCCTTCTCGG TAATAAAACC TTCCGCACGT 1750CGTTTGTTGA CGGGGCGGTT TTAGAGACTA ATGGCCCAGA GCGCCACAAT 1800 CTCTCTTTTGATGCCAGTCA GAGCACTATG GCCGCCGGCC CTTTCAGTCT 1850 CACCTATGCC GCCTCTGCTGCTGGGCTGGA GGTGCGCTAT GTCGCCGCCG 1900 GGCTTGACCA CCGGGCGGTT TTTGCCCCCGGCGTTTCACC CCGGTCAGCC 1950 CCTGGCGAGG TCACCGCCTT CTGTTCTGCC CTATACAGGTTTAATCGCGA 2000 GGCCCAGCGC CTTTCGCTGA CCGGTAATTT TTGGTTCCAT CCTGAGGGGC2050 TCCTTGGCCC CTTTGCCCCG TTTTCCCCCG GGCATGTTTG GGAGTCGGCT 2100AATCCATTCT GTGGCGAGAG CACACTTTAC ACCCGCACTT GGTCGGAGGT 2150 TGATGCTGTTCCTAGTCCAG CCCAGCCCGA CTTAGGTTTT ACATCTGAGC 2200 CTTCTATACC TAGTAGGGCCGCCACACCTA CCCCGGCGGC CCCTCTACCC 2250 CCCCCTGCAC CGGATCCTTC CCCTACTCTCTCTGCTCCGG CGCGTGGTGA 2300 GCCGGCTCCT GGCGCTACCG CCAGGGCCCC AGCCATAACCCACCAGACGG 2350 CCCGGCATCG CCGCCTGCTC TTTACCTACC CGGATGGCTC TAAGGTGTTC2400 GCCGGCTCGC TGTTTGAGTC GACATGTACC TGGCTCGTTA ACGCGTCTAA 2450TGTTGACCAC CGCCCTGGCG GTGGGCTCTG TCATGCATTT TACCAGAGGT 2500 ACCCCGCCTCCTTTGATGCT GCCTCTTTTG TGATGCGCGA CGGCGCGGCC 2550 GCCTACACAT TAACCCCCCGGCCAATAATT CATGCCGTCG CTCCTGATTA 2600 TAGGTTGGAA CATAACCCAA AGAGGCTTGAGGCTGCCTAC CGGGAGACTT 2650 GCTCCCGCCT CGGTACCGCT GCATACCCAC TCCTCGGGACCGGCATATAC 2700 CAGGTGCCGA TCGGTCCCAG TTTTGACGCC TGGGAGCGGA ATCACCGCCC2750 CGGGGACGAG TTGTACCTTC CTGAGCTTGC TGCCAGATGG TTCGAGGCCA 2800ATAGGCCGAC CTGCCCAACT CTCACTATAA CTGAGGATGT TGCGCGGACA 2850 GCAAATCTGGCTATCGAACT TGACTCAGCC ACAGACGTCG GCCGGGCCTG 2900 TGCCGGCTGT CGAGTCACCCCCGGCGTTGT GCAGTACCAG TTTACCGCAG 2950 GTGTGCCTGG ATCCGGCAAG TCCCGCTCTATTACCCAAGC CGACGTGGAC 3000 GTTGTCGTGG TCCCGACCCG GGAGTTGCGT AATGCCTGGCGCCGCCGCGG 3050 CTTCGCTGCT TTCACCCCGC ACACTGCGGC TAGAGTCACC CAGGGGCGCC3100 GGGTTGTCAT TGATGAGGCC CCGTCCCTTC CCCCTCATTT GCTGCTGCTC 3150CACATGCAGC GGGCCGCCAC CGTCCACCTT CTTGGCGACC CGAATCAGAT 3200 CCCAGCCATCGATTTTGAGC ACGCCGGGCT CGTTCCCGCC ATCAGGCCCG 3250 ATTTGGCCCC CACCTCCTGGTGGCATGTTA CCCATCGCTG CCCTGCGGAT 3300 GTATGTGAGC TAATCCGCGG CGCATACCCTATGATTCAGA CCACTAGTCG 3350 GGTCCTCCGG TCGTTGTTCT GGGGTGAGCC CGCCGTTGGGCAGAAGCTAG 3400 TGTTCACCCA GGCGGCTAAG GCCGCCAACC CCGGTTCAGT GACGGTCCAT3450 GAGGCACAGG GCGCTACCTA CACAGAGACT ACCATCATTG CCACGGCAGA 3500TGCTCGAGGC CTCATTCAGT CGTCCCGAGC TCATGCCATT GTTGCCTTGA 3550 CGCGCCACACTGAGAAGTGC GTCATCATTG ACGCACCAGG CCTGCTTCGC 3600 GAGGTGGGCA TCTCCGATGCAATCGTTAAT AACTTTTTCC TTGCTGGTGG 3650 CGAAATTGGC CACCAGCGCC CATCTGTTATCCCTCGCGGC AATCCTGACG 3700 CCAATGTTGA CACCTTGGCT GCCTTCCCGC CGTCTTGCCAGATTAGCGCC 3750 TTCCATCAGT TGGCTGAGGA GCTTGGCCAC AGACCTGCCC CTGTCGCGGC3800 TGTTCTACCG CCCTGCCCTG AGCTTGAACA GGGCCTTCTC TACCTGCCCC 3850AAGAACTCAC CACCTGTGAT AGTGTCGTAA CATTTGAATT AACAGATATT 3900 GTGCATTGTCGTATGGCCGC CCCGAGCCAG CGCAAGGCCG TGCTGTCCAC 3950 GCTCGTGGGC CGTTATGGCCGCCGCACAAA GCTCTACAAT GCCTCCCACT 4000 CTGATGTTCG CGACTCTCTC GCCCGTTTTATCCCGGCCAT TGGCCCCGTA 4050 CAGGTTACAA CCTGTGAATT GTACGAGCTA GTGGAGGCCATGGTCGAGAA 4100 GGGCCAGGAC GGCTCCGCCG TCCTTGAGCT CGACCTTTGT AGCCGCGACG4150 TGTCCAGGAT CACCTTCTTC CAGAAAGATT GTAATAAATT CACCACGGGG 4200GAGACCATCG CCCATGGTAA AGTGGGCCAG GGCATTTCGG CCTGGAGTAA 4250 GACCTTCTGTGCCCTTTTCG GCCCCTGGTT CCGTGCTATT GAGAAGGCTA 4300 TCCTGGCCCT GCTCCCTCAGGGTGTGTTTT ATGGGGATGC CTTTGATGAC 4350 ACCGTCTTCT CGGCGGCTGT GGCCGCAGCAAAGGCATCCA TGGTGTTCGA 4400 GAATGACTTT TCTGAGTTTG ATTCCACCCA GAATAATTTTTCCTTGGGCC 4450 TAGAGTGTGC TATTATGGAG GAGTGTGGGA TGCCGCAGTG GCTCATCCGC4500 TTGTACCACC TTATAAGGTC TGCGTGGATT CTGCAGGCCC CGAAGGAGTC 4550CCTGCGAGGG TTTTGGAAGA AACACTCCGG TGAGCCCGGC ACCCTTCTGT 4600 GGAATACTGTCTGGAACATG GCCGTTATCA CCCACTGTTA TGATTTCCGC 4650 GATCTGCAGG TGGCTGCCTTTAAAGGTGAT GATTCGATAG TGCTTTGCAG 4700 TGAGTACCGT CAGAGCCCAG GGGCTGCTGTCCTGATTGCT GGCTGTGGCC 4750 TAAAGTTGAA GGTGGATTTC CGTCCGATTG GTCTGTATGCAGGTGTTGTG 4800 GTGGCCCCCG GCCTTGGCGC GCTTCCTGAT GTCGTGCGCT TCGCCGGTCG4850 GCTTACTGAG AAGAATTGGG GCCCTGGCCC CGAGCGGGCG GAGCAGCTCC 4900GCCTCGCTGT GAGTGATTTT CTCCGCAAGC TCACGAATGT AGCTCAGATG 4950 TGTGTGGATGTTGTCTCTCG TGTTTATGGG GTTTCCCCTG GGCTCGTTCA 5000 TAACCTGATT GGCATGCTACAGGCTGTTGC TGATGGCAAG GCTCATTTCA 5050 CTGAGTCAGT GAAGCCAGTG CTTGACCTGACAAATTCAAT TCTGTGTCGG 5100 GTGGAATGAA TAACATGTCT TTTGCTGCGC CCATGGGTTCGCGACCATGC 5150 GCCCTCGGCC TATTTTGCTG TTGCTCCTCA TGTTTCTGCC TATGCTGCCC5200 GCGCCACCGC CCGGTCAGCC GTCTGGCCGC CGTCGTGGGC GGCGCAGCGG 5250CGGTTCCGGC GGTGGTTTCT GGGGTGACCG GGTTGATTCT CAGCCCTTCG 5300 CAATCCCCTATATTCATCCA ACCAACCCCT TCGCCCCCGA TGTCACCGCT 5350 GCGGCCGGGG CTGGACCTCGTGTTCGCCAA CCCGCCCGAC CACTCGGCTC 5400 CGCTTGGCGT GACCAGGCCC AGCGCCCCGCCGCTGCCTCA CGTCGTAGAC 5450 CTACCACAGC TGGGGCCGCG CCGCTAACCG CGGTCGCTCCGGCCCATGAC 5500 ACCCCGCCAG TGCCTGATGT TGACTCCCGC GGCGCCATCC TGCGCCGGCA5550 GTATAACCTA TCAACATCTC CCCTCACCTC TTCCGTGGCC ACCGGCACAA 5600ATTTGGTTCT TTACGCCGCT CCTCTTAGCC CGCTTCTACC CCTCCAGGAC 5650 GGCACCAATACTCATATAAT GGCTACAGAA GCTTCTAATT ATGCCCAGTA 5700 CCGGGTTGCT CGTGCCACAATTCGCTACCG CCCGCTGGTC CCCAACGCTG 5750 TTGGTGGCTA CGCTATCTCC ATTTCGTTCTGGCCACAGAC CACCACCACC 5800 CCGACGTCCG TTGACATGAA TTCAATAACC TCGACGGATGTCCGTATTTT 5850 AGTCCAGCCC GGCATAGCCT CCGAGCTTGT TATTCCAAGT GAGCGCCTAC5900 ACTATCGCAA CCAAGGTTGG CGCTCTGTTG AGACCTCCGG GGTGGCGGAG 5950GAGGAGGCCA CCTCTGGTCT TGTCATGCTC TGCATACATG GCTCACCTGT 6000 AAATTCTTATACTAATACAC CCTATACCGG TGCCCTCGGG CTGTTGGACT 6050 TTGCCCTCGA ACTTGAGTTCCGCAACCTCA CCCCCGGTAA TACCAATACG 6100 CGGGTCTCGC GTTACTCCAG CACTGCCCGTCACCGCCTTC GTCGCGGTGC 6150 AGATGGGACT GCCGAGCTCA CCACCACGGC TGCTACTCGCTTCATGAAGG 6200 ACCTCTATTT TACTAGTACT AATGGTGTTG GTGAGATCGG CCGCGGGATA6250 GCGCTTACCC TGTTTAACCT TGCTGACACC CTGCTTGGCG GTCTACCGAC 6300AGAATTGATT TCGTCGGCTG GTGGCCAGCT GTTCTACTCT CGCCCCGTCG 6350 TCTCAGCCAATGGCGAGCCG ACTGTTAAGC TGTATACATC TGTGGAGAAT 6400 GCTCAGCAGG ATAAGGGTATTGCAATCCCG CATGACATCG ACCTCGGGGA 6450 ATCCCGTGTA GTTATTCAGG ATTATGACAACCAACATGAG CAGGACCGAC 6500 CGACACCTTC CCCAGCCCCA TCGCGTCCTT TTTCTGTCCTCCGAGCTAAC 6550 GATGTGCTTT GGCTTTCTCT CACCGCTGCC GAGTATGACC AGTCCACTTA6600 CGGCTCTTCG ACCGGCCCAG TCTATGTCTC TGACTCTGTG ACCTTGGTTA 6650ATGTTGCGAC CGGCGCGCAG GCCGTTGCCC GGTCACTCGA CTGGACCAAG 6700 GTCACACTTGATGGTCGCCC CCTTTCCACC ATCCAGCAGT ATTCAAAGAC 6750 CTTCTTTGTC CTGCCGCTCCGCGGTAAGCT CTCCTTTTGG GAGGCAGGAA 6800 CTACTAAAGC CGGGTACCCT TATAATTATAACACCACTGC TAGTGACCAA 6850 CTGCTCGTTG AGAATGCCGC TGGGCATCGG GTTGCTATTTCCACCTACAC 6900 TACTAGCCTG GGTGCTGGCC CCGTCTCTAT TTCCGCGGTT GCTGTTTTAG6950 CCCCCCACTC TGTGCTAGCA TTGCTTGAGG ATACCATGGA CTACCCTGCC 7000CGCGCCCATA CTTTCGATGA CTTCTGCCCG GAGTGCCGCC CCCTTGGCCT 7050 CCAGGGTTGTGCTTTTCAGT CTACTGTCGC TGAGCTTCAG CGCCTTAAGA 7100 TGAAGGTGGG TAAAACTCGGGAGTTATAGT TTATTTGCTT GTGCCCCCCT 7150 TCTTTCTGTT GCTTATTT 7168 25 basepairs nucleic acid single linear 5 ACATTTGAAT TCACAGACAT TGTGC 25 26base pairs nucleic acid single linear 6 ACACAGATCT GAGCTACATT CGTGAG 2626 base pairs nucleic acid single linear 7 AAAGGGATCC ATGGTGTTTG AGAATG26 25 base pairs nucleic acid single linear 8 ACTCACTGCA GAGCACTATCGAATC 25 22 base pairs nucleic acid single linear 9 CGGTAAACTGGTACTGCACA AC 22 22 base pairs nucleic acid single linear 10 AAGTCCCGCTCTATTACCCA AG 22 21 base pairs nucleic acid single linear 11 ACCCACGGGTGTTGGTTTTT G 21 21 base pairs nucleic acid single linear 12 TTCTTGGGGCAGGTAGAGAA G 21 26 base pairs nucleic acid single linear 13 TTATTGAATTCATGTCAACG GACGTC 26 21 base pairs nucleic acid single linear 14AATAATTCAT GCCGTCGCTC C 21 21 base pairs nucleic acid single linear 15AAGCTCAGGA AGGTACAACT C 21 24 base pairs nucleic acid single linear 16AAATCGATGG CTGGGATCTG ATTC 24 21 base pairs nucleic acid single linear17 GAGGCATTGT AGAGCTTTGT G 21 22 base pairs nucleic acid single linear18 GATGTTGCAC GGACAGCAAA TC 22 24 base pairs nucleic acid single linear19 ATCTCCGATG CAATCGTTAA TAAC 24 21 base pairs nucleic acid singlelinear 20 TAATCCATTC TGTGGCGAGA G 21 21 base pairs nucleic acid singlelinear 21 AAGTGTGACC TTGGTCCAGT C 21 23 base pairs nucleic acid singlelinear 22 TTGCTCGTGC CACAATTCGC TAC 23 21 base pairs nucleic acid singlelinear 23 CATTTCACTG AGTCAGTGAA G 21 21 base pairs nucleic acid singlelinear 24 TAATTATAAC ACCACTGCTA G 21 21 base pairs nucleic acid singlelinear 25 GATTGCAATA CCCTTATCCT G 21 23 base pairs nucleic acid singlelinear 26 ATTAAACCTG TATAGGGCAG AAC 23 21 base pairs nucleic acid singlelinear 27 AAGTTCGATA GCCAGATTTG C 21 21 base pairs nucleic acid singlelinear 28 TCATGTTGGT TGTCATAATC C 21 21 base pairs nucleic acid singlelinear 29 GATGACGCAC TTCTCAGTGT G 21 19 base pairs nucleic acid singlelinear 30 AGAACAACGA ACGGAGAAC 19 21 base pairs nucleic acid singlelinear 31 AGATCCCAGC CATCGACTTT G 21 21 base pairs nucleic acid singlelinear 32 TAGTAGTGTA GGTGGAAATA G 21 21 base pairs nucleic acid singlelinear 33 GTGTGGTTAT TCAGGATTAT G 21 21 base pairs nucleic acid singlelinear 34 ACTCTGTGAC CTTGGTTAAT G 21 21 base pairs nucleic acid singlelinear 35 AACTCAAGTT CGAGGGCAAA G 21 21 base pairs nucleic acid singlelinear 36 CGCTTACCCT GTTTAACCTT G 21 24 base pairs nucleic acid singlelinear 37 ATCCCCTATA TTCATCCAAC CAAC 24 21 base pairs nucleic acidsingle linear 38 CTCCTCATGT TTCTGCCTAT G 21 22 base pairs nucleic acidsingle linear 39 GCCAGAACGA AATGGAGATA GC 22 21 base pairs nucleic acidsingle linear 40 CTCAGACATA AAACCTAAGT C 21 21 base pairs nucleic acidsingle linear 41 TGCCCTATAC AGGTTTAATC G 21 19 base pairs nucleic acidsingle linear 42 ACCGGCATAT ACCAGGTGC 19 21 base pairs nucleic acidsingle linear 43 ACATGGCTCA CTCGTAAATT C 21 21 base pairs nucleic acidsingle linear 44 AACATTAGAC GCGTTAACGA G 21 21 base pairs nucleic acidsingle linear 45 CTCTTTTGAT GCCAGTCAGA G 21 22 base pairs nucleic acidsingle linear 46 ACCTACCCGG ATGGCTCTAA GG 22 25 base pairs nucleic acidsingle linear 47 TATGGGAATT CGTGCCGTCC TGAAG 25 21 base pairs nucleicacid single linear 48 AGTGGGAGCA GTATACCAGC G 21 21 base pairs nucleicacid single linear 49 CTGCTATTGA GCAGGCTGCT C 21 21 base pairs nucleicacid single linear 50 GGGCCATTAG TCTCTAAAAC C 21 19 base pairs nucleicacid single linear 51 GAGGTTTTCT GGAATCATC 19 15 base pairs nucleic acidsingle linear 52 GCATAGGTGA GACTG 15 18 base pairs nucleic acid singlelinear 53 AGTTACAGCC AGAAAACC 18 33 base pairs nucleic acid singlelinear 54 CCATGGATCC TCGGCCTATT TTGCTGTTGC TCC 33 18 base pairs nucleicacid single linear 55 AGGCAGACCA CATATGTG 18 20 base pairs nucleic acidsingle linear 56 GGTGCACTCC TGACCAAGCC 20 19 base pairs nucleic acidsingle linear 57 ATTGGCTGCC ACTTTGTTC 19 21 base pairs nucleic acidsingle linear 58 ACCCTCATAC GTCACCACAA C 21 24 base pairs nucleic acidsingle linear 59 GCGGTGGACC ACATTAGGAT TATC 24 19 base pairs nucleicacid single linear 60 CATGATATGT CACCATCTG 19 19 base pairs nucleic acidsingle linear 61 GTCATCCATA ACGAGCTGG 19 33 base pairs nucleic acidsingle linear 62 AGCGGAATTC GAGGGGCGGC ATAAAGAACC AGG 33 36 base pairsnucleic acid single linear 63 GCGCTGAATT CGGATCACAA GCTCAGAGGC TATGCC 3630 base pairs nucleic acid single linear 64 GTATAACGGA TCCACATCTCCCCTTACCTC 30 30 base pairs nucleic acid single linear 65 TAACCTGGATCCTTATGCCG CCCCTCTTAG 30 38 base pairs nucleic acid single linear 66AAATTGGATC CTGTGTCGGG TGGAATGAAT AACATGTC 38 37 base pairs nucleic acidsingle linear 67 ATCGGCAGAT CTGATAGAGC GGGGACTTGC CGGATCC 37 28 basepairs nucleic acid single linear 68 TACCCTGCCC GCGCCCATAC TTTTGATG 28 33base pairs nucleic acid single linear 69 GGCTGAGATC TGGTTCGGGTCGCCAAGAAG GTG 33 27 base pairs nucleic acid single linear 70 TACAGATCTATACAACTTAA CAGTCGG 27 29 base pairs nucleic acid single linear 71GCGGCAGATC TCACCGACAC CATTAGTAC 29 28 base pairs nucleic acid singlelinear 72 CCGTCGGATC CCAGGGGCTG CTGTCCTG 28 31 base pairs nucleic acidsingle linear 73 AAAGGAATTC AAGACCAGAG GTAGCCTCCT C 31 28 base pairsnucleic acid single linear 74 GTTGATATGA ATTCAATAAC CTCGACGG 28 36 basepairs nucleic acid single linear 75 TTTGGATCCT CAGGGAGCGC GGAACGCAGAAATGAG 36 26 base pairs nucleic acid single linear 76 TCACTCGTGAATTCCTATAC TAATAC 26 34 base pairs nucleic acid single linear 77TTTGGATCCT CAGGGAGCGC GGAACGCAGA AATG 34 25 base pairs nucleic acidsingle linear 78 TGATAGAGCG GGACTTGCCG GATCC 25 24 base pairs nucleicacid single linear 79 TTGCATTAGG TTAATGAGGA TCTC 24 25 base pairsnucleic acid single linear 80 ACCTGCTTCC TTCAGCCTGC AGAAG 25 29 basepairs nucleic acid single linear 81 GCGGTGGATC CGCTCCCAGG CGTCAAAAC 2929 base pairs nucleic acid single linear 82 GGGCGGATCG AATTCGAGACCCTTCTTGG 29 27 base pairs nucleic acid single linear 83 AGGATGGATCCATAAGTTAC CGATCAG 27 29 base pairs nucleic acid single linear 84GGCTGGAATT CCTCTGAGGA CGCCCTCAC 29 27 base pairs nucleic acid singlelinear 85 GCCGAAGATC TATCGGACAT AGACCTC 27 30 base pairs nucleic acidsingle linear 86 CAGACGACGG ATCCCCTTGG ATATAGCCTG 30 40 base pairsnucleic acid single linear 87 GGCCGAATTC AGGCAGACCA CATATGTGGTCGATGCCATG 40 25 base pairs nucleic acid single linear 88 GCAGGTGTGCCTGGATCCGG CAAGT 25 30 base pairs nucleic acid single linear 89GTTAGAATTC CGGCCCAGCT GTGGTAGGTC 30 24 base pairs nucleic acid singlelinear 90 CCGTCCGATT GGTCTGTATG CAGG 24 22 base pairs nucleic acidsingle linear 91 TACCAGTTTA CTGCAGGTGT GC 22 22 base pairs nucleic acidsingle linear 92 CAAGCCGATG TGGACGTTGT CG 22 24 base pairs nucleic acidsingle linear 93 GGCGCTGGGC CTGGTCACGC CAAG 24 22 base pairs nucleicacid single linear 94 GCAGAAACTA GTGTTGACCC AG 22 22 base pairs nucleicacid single linear 95 TAGGTCTACG ACGTGAGGCA AC 22 21 base pairs nucleicacid single linear 96 TACAATCTTT CAGGAAGAAG G 21 21 base pairs nucleicacid single linear 97 CCCACACTCC TCCATAATAG C 21 24 base pairs nucleicacid single linear 98 GATAGTGCTT TGCAGTGAGT ACCG 24 30 base pairsnucleic acid single linear 99 GTATAACGGA TCCACATCTC CCCTTACCTC 30 27base pairs nucleic acid single linear 100 TACAGATCTA TACAACTTAA CAGTCGG27 29 base pairs nucleic acid single linear 101 GCGGCAGATC TCACCGACACCATTAGTAC 29 30 base pairs nucleic acid single linear 102 TAACCTGGATCCTTATGCCG CCCCTCTTAG 30 36 base pairs nucleic acid single linear 103GCACAACCTA GGTTACTATA ACTCCCGAGT TTTACC 36 33 base pairs nucleic acidsingle linear 104 GGGTTCCCTA GGATGCGCCC TCGGCCTATT TTG 33 33 base pairsnucleic acid single linear 105 CGTGGGCCTA GGAGCGGCGG TTCCGGCGGT GGT 3333 base pairs nucleic acid single linear 106 GCTTGGCCTA GGCAGGCCCAGCGCCCCGCC GCT 33 33 base pairs nucleic acid single linear 107CCGCCACCTA GGGATGTTGA CTCCCGCGGC GCC 33

What is claimed is:
 1. A nucleic acid molecule encoding an amino acidsequence according to SEQ ID NO:2.
 2. The nucleic acid molecule of claim1, wherein said molecule has a nucleotide sequence consisting ofnucleotides 5147 to 7126 of SEQ ID NO:4.
 3. A recombinant vectorcomprising the nucleic acid molecule of claim
 1. 4. The recombinantvector of claim 3, wherein said vector is a baculovirus vector.
 5. Arecombinant vector comprising the nucleic acid molecule of claim
 2. 6.The recombinant vector of claim 5, wherein said vector is a baculovirusvector.
 7. The vector of claim 6, wherein said vector has ATCC accessionnumber
 75299. 8. A host cell containing the vector of claim
 3. 9. A hostcell containing the vector of claim
 4. 10. A host cell containing thevector of claim
 5. 11. A host cell containing the vector of claim
 6. 12.A host cell containing the vector of claim 7.